research article cross-platform evaluation of commercial real-time sybr green rt-pcr...
TRANSCRIPT
Research ArticleCross-Platform Evaluation of Commercial Real-TimeSYBR Green RT-PCR Kits for Sensitive and Rapid Detectionof European Bat Lyssavirus Type 1
Evelyne Picard-Meyer Carine Peytavin de Garam Jean Luc Schereffer Clotilde MarchalEmmanuelle Robardet and Florence Cliquet
Anses Laboratory for Rabies and Wildlife WHO Collaborating Centre for Research and Management in Zoonoses ControlOIE Reference Laboratory for Rabies European Union Reference Laboratory for Rabies European Union Reference Laboratoryfor Rabies Serology Technopole Agricole et Veterinaire CS 40009 54220 Malzeville France
Correspondence should be addressed to Evelyne Picard-Meyer evelynepicard-meyeransesfr
Received 3 October 2014 Accepted 12 January 2015
Academic Editor Cheng-Feng Qin
Copyright copy 2015 Evelyne Picard-Meyer et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCRkits using real-time PCR assays Two real-time thermocyclers showing different throughput capacities were used The analysedperformance evaluation criteria included the generation of standard curve reaction efficiency analytical sensitivity intra- andinterassay repeatability as well as the costs and the practicability of kits and thermocycling times We found that the optimisedone-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used while nodifference was detected in reaction efficiency R2 values and intra- and interreproducibility between the two methods The limit ofdetection at the 95 confidence level varied between 15 to 981 copies120583L and 41 to 171 for one-step kits and two-step kits respectivelyOf the ten kits tested the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95 of confidenceof 20 and 22 copies120583L on the thermocyclers Rotor gene Q MDx and MX3005P respectively The study demonstrated the pivotalinfluence of the thermocycler on PCR performance for the detection of rabies RNA as well as that of the master mixes
1 Introduction
Rabies which is caused by all members of the genus Lyssavi-rus family Rhabdoviridae is a viral encephalitis that isfound on every continent except Antarctica The Lyssavirusgenus is divided into 14 species the majority of which arepredominantly associated with infection in bats [1] Bats arenatural reservoirs for all lyssaviruses and are considered torepresent the ancestral host for the lyssaviruses [2] In Europebat rabies is caused by four lyssaviruses namely Europeanbat Lyssavirus type 1 (EBLV-1) European bat Lyssavirus type2 (EBLV-2) Bokeloh bat Lyssavirus (BBLV) and Lleida batLyssavirus (LLEBV) Between 1977 and 2013 more than 1000cases of bat rabieswere reported [3] with themajority (gt97)being attributed to EBLV-1
Rabies diagnosis based upon clinical presentation orgross pathognomonic lesions is unreliable because signsof the disease are not characteristic and may vary greatlyfrom one animal to another Therefore diagnosis in ani-mals relies on postmortem laboratory findings The WHOldquogold standardrdquo test for rabies diagnosis in animals is thefluorescent antibody test (FAT) in which FITC anti-rabiesvirus antibody is applied to selected regions of the braincollected postmortem [4] Evidence of a rabies infection canbe demonstrated through the detection of antigen (FAT)infectious virus (rabies tissue culture infection test (RTCIT)mouse inoculation test (MIT)) or viral RNA (nucleic acidamplification tests) these conventional OIE diagnostic testsbeing entirely dependent on the nature and quality of thesample supplied With the advent of molecular biology since
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 839518 18 pageshttpdxdoiorg1011552015839518
2 BioMed Research International
the 1980s and due to the immense versatility and sensitivityof PCR diagnostic methods based on PCR technology havebeen extensively applied to many infectious diseases [5]To date real-time PCR is the state-of-the-art technologyfor the quantification of nucleic acids Various nucleic aciddetection-based assays have thus been published for thedetection of Lyssavirus fragments using conventional gel-based PCR NASBA or real-time PCR [6] In Europe mostlaboratories working on rabies employ conventional andorreal-time PCR [7] due to their high specificity and sensitivityThe conventional methods are reported to be very sensitivewhen using a heminested RT-PCR [8] and compared to hem-inested RT-PCR real-time PCR is shown to be about 100ndash1000-fold more sensitive [9] However due to this high PCRsensitivity which engenders a risk of sample cross contamina-tion and therefore a risk of potential false positive results RT-PCR is not recommended by the World Health Organizationfor routine postmortem diagnosis of rabies in animals [4]
Real-time RT-PCR which provides rapid sensitive andspecific detection of rabies virus decreases the risk of samplecontamination Various real-time assays have thus beendescribed since 2004 [10] for the rapid detection of RABVor other Lyssavirus species [11 12] using the one-step method[13 14] or the two-step method [12 15 16] Two systems ofreal-time assays are widely used for the detection of RABVand lyssaviruses the SYBR Green real-time RT-PCR andthe TaqMan real-time RT-PCR which is to date the mostcommon technique implemented in the laboratories workingon rabies [17]
As real-time PCR assays have become routine in diagnos-tic laboratories and because of their prominence there hasbeen rapid growth of commercially available reagent systemsand detection platforms which greatly increase the optionsavailable to laboratories In this study we evaluated severalcommercial master mixes in order to identify those whichproduce the best results using one-step and two-step real-time RT-PCR for the determination of EBLV-1 Five differentsystems for the generation of cDNA five two-step PCR kitsand five one-step qRT-PCR kits were comparedThe analysedperformance evaluation criteria included the generation ofa standard curve reaction efficiency analytical sensitivityintra- and interassay repeatability as well as the costs and thepracticability of kits and thermocycling times The goal ofthis study was also to determine if the one-step and two-stepsystems had the same reaction efficiencies for the detectionof EBLV-1 and if one system was more sensitive than anotherusing two real-time thermocyclers
2 Materials and Methods
21 Rabies Virus Isolates and RNA Extraction The Europeanbat Lyssavirus type 1 virus (EBLV-1) was selected for this studyfor the generation of in vitroRNA transcriptThe virus EBLV-1 (ANSES strain numbered 05-09) was isolated in France in2000 from a rabid bat (Eptesicus serotinus) The isolate wasmouse passaged before RNA extraction
Viral RNAwas extracted from 200120583L of 10mouse braintissue suspension using the Iprep Pure Link Virus kit (Invit-rogen France) according to the manufacturerrsquos instructions
The RNA concentration was determined spectrophotometri-cally and the RNA was stored at minus70∘C until use
22 Preparation of In Vitro RNA Transcripts Briefly a one-step RT-PCR amplification was performed as previouslydescribed [8] on 5 120583L of extracted RNA (ie 250ndash500 ng)with specific rabies primers (JW12 (forward) 51015840-ATGTAA-CACCYCTACAATG and JW6 (reverse) 51015840-CARTTVGCR-CACATYTTRTG) The 606 bp PCR products were purifiedand inserted into a pGEM T easy vector (Promega France)according to the manufacturerrsquos instructions Recombinantplasmids were purified using the PerfectPrep EndoFreePlasmid Maxi Kit (5Prime France) and then concentratedusing a step of sodium acetateethanol precipitation and lin-earised with the PstI restriction enzyme (Promega France)The clones of PCR products were bidirectionally sequencedwith sense and antisense primers T7 and SP6 by BeckmanCoulter Genomics (Takeley United Kingdom) to confirm thepresence of the target insert
In vitro transcription was performed using T7 RNApolymerase according to the manufacturerrsquos instructions(MaxiScript SP6T7 kit Ambion France)The resulting RNAtranscripts were purified as recommended by the manufac-turer using 2120583L of DNAse I during 15min at 37∘C followedby a final purification step using the RNeasymini kit (QiagenFrance) The RNA concentration was determined by usingthe Qubit Fluorometer (Invitrogen France) RNA transcriptswere aliquoted to 107 copies120583L and then stored at minus70∘CTheRNAmolecule copy numberwas calculated following themanufacturerrsquos instructions (MaxiScript SP6T7 kit AmbionFrance) using the following formula 119884 molecules120583L =(119883 g120583L RNA[transcript length in nucleotides times 330] times6022sdot1023) The estimated number of copies was of 268 sdot 1011for EBLV-1 RNA transcript
The quality of RNA transcript was checked on serialtenfold dilutions (106 to 1 copie120583L) through measurementsof efficiency of amplification using a two-step SYBR GreenRT-PCR [12] PCR efficiency was of 99
23 Real-Time RT-PCR Assays
231 Primers Used Universal pan Lyssavirus primers wereselected for this study Two questionnaires respectivelyundertaken in 2011-2012 by the EURL laboratory havedemonstrated that 13 out of 21 National Reference Laborato-ries for Rabies (NRLs) used primers JW12 and N165-146 pre-viously described by Wakeley et al [18] These specific rabiesprimers (JW12 (forward) ATGTAACACCYCTACAATGand N165-146 (reverse) GCAGGGTAYTTRTACTCATA)were designed to detect the rabies virus N gene of all knownlyssaviruses [12]
232 Two-Step RT-qPCR Assays
(1) Commercial Master Mix Kits
(a) Reverse Transcription Kits (Table 1(a)) Five differentreverse transcription (RT) systems provided by five differentcompanies and five different qPCR kits provided by the same
BioMed Research International 3
Table 1 Characteristics of the commercial master mix kits used In (a) the reactions mix and thermocycling conditions for the five RT kitsare detailed in (b) the reactions mix and thermocycling conditions for the five qPCR SybrGreen kits (two-step) and in (c) the reactions Mixand thermocycling conditions for the five qRT-PCR SybrGreen kits (one-step) used in this study
(a) Reactions mix and thermocycling conditions for the five RT kits
Manufacturer Reversetranscription kit RTase and primers Thermocycling
conditions Reaction mixlowastlowast
Bio-Rad Iscript select cDNAsynthesis kit
M-MLV RTlowastRandom primer
5min-25∘C30min-42∘C5min-85∘C
4 120583L Iscript Mix (5x) + 2120583L Random primer +1 120583L Iscript RTase + 8 120583L RNase free water
QiagenQuantiTectReverse
Transcription Kit
Quantiscript RTaseMix OligodT
+ random primer
2min-42∘C30min-42∘C3min-95∘C
2120583L gDNAWipeout Buffer (7x) + 4 120583LQuantiscript RT Buffer + 1120583L QuantiscriptRTase + 1 120583L RT primer + 7 120583L RNase free
water
FermentasMaxima HMinusFirst Strand cDNA
Synthesis Kit
M-MLV RTlowastRandom primer
5min-65∘C10min-25∘C30min-50∘C5min-85∘C
4120583L RT Buffer (5x) + 1 120583L Random primer +1 120583L dNTP (10mM) + 1 120583L Maxima HMinus
Ez Mix + 8 120583L RNase free water
Invitrogen SuperScript ViloMaster Mix
M-MLV RTlowastRandom primer
10min-25∘C30min-42∘C5min-85∘C
4120583L SuperScript Vilo MasterMix (5x) + 11 120583LRNase free water
Quanta qScript cDNASynthesis Kit
M-MLV RTlowastMix OligodT
+ random primer
5min-22∘C30min-42∘C5min-85∘C
4 120583L qScript Reaction Mix (5x) + 1 120583L qScriptRTase + 10 120583L RNase free water
lowastMoloney Murine Leukemia ViruslowastlowastTotal volume of reaction is 20120583L containing 5120583L of RNA sample and 15 120583L of reaction mix All RT assays were performed on the Mastercycler Eppendorfthermal cyclerRTase reverse transcriptase
(b) Reactions mix and thermocycling conditions for the five qPCR SybrGreen kits (two-step)
Manufacturer qPCRSybrGreen kit Thermocycling conditions Reaction mixlowast
Bio-RadSsoFastEvaGreenSupermix
30 sec-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 20 120583L containing 2 120583L of cDNA and18 120583L of a reaction mix (1x Sso Fast master mix035 120583M [05 120583M] each of pan-Lyssavirus primers)
QiagenQuantiTectSYBR GreenPCR Kit
15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 25 120583L containing 2 120583L of cDNA and23 120583L of reaction mix (1x Quantitect Master mix05 120583M [06 120583M] each of pan-Lyssavirus primers)
FermentasMaxima SYBRGreen qPCRMaster Mix
10min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 25 120583L containing 2 120583L of cDNA and23 120583L of reaction mix (1x Maxima SYBR Green mastermix 04 120583M [1 120583M] each of pan-Lyssavirus primers)
Invitrogen
Express SYBRGreenER qPCR
SupermixUniversal
2min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 20 120583L containing 2 120583L of cDNA and18 120583L of reaction mix (1x Express SYBR GreenERSupermix 04 120583M [2 120583M] each of pan-Lyssavirus
primers) [50 nM of ROX dye]
Quanta Perfecta SYBRGreen SuperMix
3min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 25 120583L containing 2 120583L of cDNA and23120583L of reaction mix (1x Perfecta SYBR GreenSupermix 03 120583M [1 120583M] each of pan-Lyssavirus
primers)lowastThe PCR reaction protocol corresponds to PCR assays performed on the Rotor Gene Q MDx The concentrations of primers used on the real timethermocycler Mx3005P are shown in brackets [ ]
(c) Reactions mix and thermocycling conditions for the five qRT-PCR SYBR Green kits (one-step)
Manufacturer qRT-PCRSybrGreen kit Thermocycling conditions Reaction mixlowast
Qiagen QuantiTect SYBRGreen RT-PCR Kit
30min-50∘C 15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x QuantiTect SYBR Green master mix1 Quantitect RT mix 06 120583M [2120583M]
each of pan-Lyssavirus primers
4 BioMed Research International
(c) Continued
Manufacturer qRT-PCRSybrGreen kit Thermocycling conditions Reaction mixlowast
QiagenRotor Gene-1 stepRotor-Gene SYBRGreen RT-PCR Kit
30min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x Rotor-Gene master mix 1Rotor-Gene RT mix 1 120583M [2 120583M] each of
pan-Lyssavirus primers
Fermentas Verso 1-stepQRT-PCR Kit
30min-50∘C 15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x Verso SYBR Green master mix 5 RTenhancer 1 Verso enzyme mix 02 120583M[03 120583M] each of pan-Lyssavirus primers
Invitrogen
SuperScript IIIPlatinum SYBRGreen One-StepqRT-PCR Kit
10min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x SuperScript III Platinium master mix2 enzyme mix 03 120583M [05120583M] each of
pan-Lyssavirus primers
QuantaqScript One-StepSYBR GreenRT-qPCR Kit
30min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x qScript master mix 2 qScript enzymemix 04 120583M [02 120583M] each of
pan-Lyssavirus primerslowastTotal volume of reaction is 25 120583L containing 2120583L of RNA sample and 23120583L of reactionmixThe PCR reaction protocol corresponds to PCR assays performedon the Rotor Gene Q MDx The concentrations of primers used on the real time thermocycler Mx3005P are shown in brackets [ ]
five suppliers were tested with the two-step method Thefollowing RT kits were compared Iscript (IScript cDNA syn-thesis kit Bio-Rad France) QuantiTect (Quantitect Reversetranscription kit Qiagen France) Maxima (Maxima HMinus First Strand cDNA synthesis kit Fermentas France)SS Vilo (Superscript Vilo Master Mix Invitrogen France)and qScript (qScript cDNA Synthesis kit Quanta France)
(b) Two-Step SYBR Green PCR Kits (Table 1(b)) Differenttwo-step qPCR kits were tested SsoFast (SsoFast EvagreenSuper mix kit Bio-Rad France) QuantiTect (QuantiTectSYBR Green PCR kit Qiagen France) Maxima (MaximaSYBR Green qPCR master mix Fermentas France) Express(Express SYBR GreenER qPCR Supermix universal Invitro-gen France) and Perfecta (Perfecta SYBR Green SupermixQuanta France)
(2) Two-Step RT-qPCR Analysis Seven 1 10 serial dilutions(ie 106 to 1 copie120583L) of the in vitro EBLV-1 RNA transcriptwere prepared in RNAse free water then subjected to reversetranscription using the five different RT master mixes AllRT kits were optimised with regard to the time and the tem-perature of incubation (Table 1(a)) Complementary cDNAswere generated from 5120583L of each dilution of EBLV-1 RNArandomprimer oligo dT or amix of oligo dTrandomprimerincluded in each tested kit according to the recommendationsof themanufacturersThe cycling conditions and the reactionprotocol for each tested RT kit are detailed in Table 1(a)Ther-mocycling was performed on the Mastercycler Eppendorfthermal cycler (Eppendorf France)
Real-time PCRs were run on the thermocyclers RotorGene QMDx (Qiagen France) and Mx3005P (Agilent Tech-nologies France) The concentrations of the reverse (N165-146) and forward (JW12) primers were optimised for eachtested kit At least three different concentrations were exam-ined that is the one recommended by the manufacturerone below and one above the recommended primer concen-tration using the qPCR conditions described in Table 1(b)The optimised concentration of primers is recorded for each
tested qPCR kits in Table 1(b) Reactions were performed intriplicate in a total volume of 20 120583L or 25 120583L following themanufacturerrsquos instructions (Table 1(b))
The following thermal profile was used PCR initialactivation step at 95∘C for 30 sec (SsoFast) 15min (Quan-tiTect) 10min (Maxima) 2min (Express) or 3min (Per-fecta) according to themanufacturerrsquos recommendationsThecycler conditions performed as previously described byHay-man et al [19] were the same for all assays 45 cycles of three-step cycling consisting of denaturation at 94∘C for 30 secannealing at 55∘C for 30 sec and extension at 72∘C for 30 sec
233 One-Step qRT-PCR Assays
(1) Commercial qRT-PCR Kits (Table 1(c)) Five commer-cial one-step kits provided by four suppliers were testedQuantiTect-1 step (QuantiTect SYBR Green RT-PCR kitQiagen France) RotorGene-1 step (Rotor-Gene SYBRGreenRT-PCR kit Qiagen France) Verso-1 step (Verso SYBRGreen one-step qRT-PCR kit Fermentas France) SSIIIPlatinium-1 step (SuperScript III Platinium SYBR GreenOne-step qRT-PCR kit Invitrogen France) and qScript-1step (qScript One step SYBR Green QRT-PCR kit QuantaFrance)
(2) One-Step qRT-PCR Analysis To compare the five mastermixes the assays were performed in triplicate using seventenfold dilutions (ie 106 to 1 copies120583L) of EBLV-1 RNAPCR was performed using a total volume of 25 120583L containing2 120583L of serial dilutions of EBLV-1 RNA and the master mixcomponents (119881 = 23 120583L) from each representative kit whichwere prepared according to the respective manufacturerrsquosinstructions (Table 1(c)) The concentrations of the reverse(N165-146) and forward (JW12) primers were optimised foreach tested kit (Table 1(c)) At least three different con-centrations were examined the one recommended by themanufacturer one below and one above the recommendedprimer concentration (Table 1(b))
BioMed Research International 5
Real-time RT-PCR assays were performed on the ther-mocycler Rotor Gene Q MDx and Mx3005P using the samethermocycling conditions for the five kits (ie 45 cycles of30 sec-94∘C 30 sec-55∘C and 30 sec-72∘C) For each testedkit the DNA polymerase activation step was performed asrecommended by the supplier (Table 1(c))
234 Data Collection and Analysis PCR efficiency and coef-ficient of determination (1198772) values were calculated by thesoftware Rotor Gene Q Series and Mx3005Pro respectively
Negative and positive controls were included in eachassay in which a threshold setting (Ct) of 003 was used as thereference for the Rotor Gene QMDx thermocycler A thresh-old setting of 300 was used as the reference for the Mx3005Pthermocycler for all tested kits except for one two-step kit(Express) for which a threshold setting of 005 was used
24 Determination of the Performance of the Method Theperformance of the method was determined for all testedkits following OIE recommendations [20] and those of theFrench Agency for Standard Operating Procedure for PCRexperimental design [21] Performance testingwas conductedfor the two real-time platforms
241 Determination of PCR Efficiency A 106 to 103 copies120583Ldilution range corresponds to the linear dynamic range forthe ten tested kits To determine the PCR efficiency foreach tested kit a standard curve was plotted based on Ctvalues obtained from samples containing 106 to 103 copies120583Lagainst the log of the estimated RNA copy number in thesample The coefficient of determination (1198772) was calculatedand considered as suitable when not lower than 099 Theslope of the standard curves was used for the determinationof PCR efficiency (119864 = 10[minus1slope] minus 1) The efficiency wasconsidered satisfactory when it is not lower than 90 and notabove 110 [22]
242 Determination of the Limit of Detection at 95 Thelimit of detection at 95 (LOD 95) which corresponds tothe lowest RNA concentration that could be detected with95 confidence was calculated using the inverse standardnormal distribution calculation [21] The LOD 95 wascalculated using the Probit analysis based on the results ofthree runs of eight repetitions of six dilutions each of EBLV-1RNA in RNase-free water (119899 = 144 samples tested) The sixdilutions (cDNA and RNA) were selected surrounding thelimit of detection of real-time PCR assays
On the Rotor Gene Q MDx gene cycler 103 to 25 cop-iessample were tested with the kit Rotor Gene-1 step 103to 50 copiessample for qScript-1 stepSSIII Platinium-1 step500 to 10 copiessample for Verso-1 step 50 to 1 copiessamplefor Quantitect-1 step 250 to 5 copiessample for Perfecta 250to 5 copiessample for QuantiTect 102 to 25 copiessamplefor Maxima 100 to 05 copiessample for Sso Fast and 100 to05 copiessample for Express
On Mx3005P cycler 2500 to 100 copiessample weretested with the kit qScript-1 step 250 to 5 copiessample forSSIII Platinium-1 stepVerso-1 step 50 to 1 copiessample forRotor Gene-1 stepQuantitect-1 step 500 to 10 copiessample
for QuantiTect 250 to 5 copies120583L for MaximaSso FastPer-fecta 250 to 5 copiessample for Perfecta and 100 to 25 cop-iessample for Express
Three independent assays were undertaken under identi-cal laboratory conditions
243 Evaluation of Reproducibility To evaluate the repro-ducibility of the ten kits inter- and intraspecific assay repro-ducibility were assessed using the coefficient of variation(CV) of theCt values for the lowest RNAconcentration show-ing 100 positive results The intra-assay repeatability wasdetermined using three independent runs of eight replicatesof EBLV-1 (119899 = 24 samples tested) The interassay repeata-bility was assessed for the same serial replicates in threeindependent assays using identical laboratory conditionsCVs expressed as percentages were obtained by dividing thestandard deviation of each tested sample by the mean of thecycle threshold values and multiplying that result by 100
3 Results
31 Differences in RNADetection Levels Using Two-StepAssays
311 Reverse Transcription Kits Of the five reverse transcrip-tion kits tested in combination with the SSoFast kit for cDNAamplification the SuperScript Vilo yielded greater sensitivityand resulted in a detection limit of 10 copies120583L (3 positivesamples out of 3 tested) The efficiency of amplification wasof 98The four other RT kits (ie kits Quantitect MaximaSS Vilo and qScript) resulted in a detection of 100 copies120583LThe amplification efficiency varied between 98 and 104The 119910-intercept for all reverse transcription kits tested incombination with the qPCR SsoFast kit varied between 3513and 3746
312 Two-Step PCR Kits All optimised two-step qPCRassays tested in combination with the Iscript RT showed sim-ilar sensitivity and enabled detection of at least 100 copies120583Lof EBLV-1 on the Rotor Gene Q MDx gene cycler Theefficiency of amplificationwhichwas satisfactory for all testedkits varied between 90 (ie kit Perfecta) and 99 (ie kitsSSo Fast Quantitect SG Maxima SG and Express) with an119910-intercept varying between 3603 and 3857
313 Comparison of Two-Step RT-qPCR Kits Provided bythe Same Manufacturer Table 2 details the results of thecomparison of the five two-step kits
(i) Rotor Gene Q MDx Of the five PCR kits tested incombination with the reverse transcriptases provided by thesame manufacturer one RT-qPCR assay (SuperScript Vilo incombination with qPCR Express kit) yielded greater sensitiv-ity and showed a detection of at least 10 copies120583L (Table 2)All amplicons investigated with the two-step method usingqPCR kits and RT kits from the same provider showedsatisfactory efficiency of amplification (100 plusmn 10) The 119910-intercept for all tested kits varied between 3473 and 3955
(ii) Mx3005P Of the five tested RT-qPCR kits one RT-qPCRassay (Maxima RTqPCRMaxima) yielded greater sensitivity
6 BioMed Research International
Table2Com
paris
onof
RT-qPC
RSybrGreen
kits(tw
o-ste
p)provided
bythes
amem
anufacturerRe
sults
offived
ifferentq
PCRkitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1RN
A
Testingof
different
RT-qPC
Rkits(tw
o-ste
p)provided
bythes
amem
anufacturerlowastlowastlowastlowast
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
IScriptR
T+qP
CRSsoF
ast
QuantiTectR
T+qP
CRQuantitect
Maxim
aRT+qP
CRMaxim
aSS
ViloRT
+qP
CREx
press
qScriptR
T+qP
CRPerfe
cta
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
5010
51658plusmn013
077
1947plusmn007
034
1650plusmn015
092
1501plusmn
005
035
1609plusmn003
019
1896plusmn004
021
2279plusmn026
115
1747plusmn009
052
1749plusmn010
054
1799plusmn029
159
105
5010
41993plusmn009
045
2289plusmn010
042
1950plusmn006
033
1821plusmn
005
026
1979plusmn003
013
2217plusmn006
025
2594plusmn009
035
2116plusmn029
138
2095plusmn003
015
2127plusmn015
072
104
5010
32328plusmn005
020
2589plusmn006
025
2295plusmn028
123
2168plusmn017
076
2318plusmn016
067
2575plusmn005
018
2960plusmn042
143
2429plusmn029
039
2440plusmn012
049
2455plusmn008
033
103
5010
22661plusmn
021
080
2964plusmn032
107
2643plusmn026
098
2481plusmn
005
019
2694plusmn008
030
2916plusmn023
080
3210plusmn019
060
2778plusmn030
109
2803plusmn074
263
2784plusmn012
042
102
5010
13045plusmn013
042
3220plusmn041
126
3059plusmn045
146
2833plusmn064
225
3140plusmn053
168
3238plusmn061
189
3512plusmn010
028
3046plusmn092
303
3105plusmn038
123
3135plusmn070
222
101
5010
023lowastlowast
13lowastlowast
23lowastlowast
3152plusmn13
3420
13lowastlowast
13lowastlowast
13lowastlowast
3276plusmn070
212
13lowastlowast
23lowastlowast
105
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
Efficiency
99
99
100
101
90
96
107
97
93
102
1198772
0999
0997
0997
0999
0999
099
9099
1099
7099
4099
8
119910-in
tercept
3665
3955
3631
3473
3767
3938
4183
3801
3850
3801
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883
num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 7
and showed a detection of at least 10 copies120583L All RT-qPCRassays showed satisfactory efficiency varying between 93 and107 with detection of at least 100 copies120583LThe 119910-interceptvaried between 3801 and 4183 for all tested kits
32 Differences in RNA Detection between the Five Kits TestedUsing the One-Step Method Table 3 details the results of thecomparison of the five one-step kits
(i) Rotor Gene Q MDx Three optimised qRT-PCR assaysrevealed poor sensitivity and enabled detection of at least103 copies120583L Of the five kits tested two kits (QuantiTect-1 step and Verso-1 step) yielded greater sensitivity with thedetection of at least 10 copies120583L (3 positive samples out of3 tested) All kits showed a satisfactory efficiency varyingbetween 94 and 104The119910-intercept varied between 3278and 3767 for the five tested one-step kits
A high presence of Primer-Dimer was shown for lowRNA concentrations for three kits SSIII Platinium-1 stepVerso-1 step and qScript-1 step
(ii) Mx3005P Of the five one-step kits tested using theMx3005P cycler three optimised one-step kits (ie Quanti-tect-1 step Rotor Gene-1 step and SSIII Platinium-1 step)revealed good sensitivity with the detection of at least10 copies120583L The two other optimised one-step assays (ieVerso-1 step and qScript-1 step) revealed poor sensitivitywith a detection of 102 and 103 copies120583L respectively Theamplification efficiencywas satisfactory for all kits (94ndash100)except for the qScript one-step kit (efficiency of 87) The 119910-intercept varied between 3680 and 4074 for all tested kitsexcept for the qScript kit (Intercept of 4074)
33 Comparison of Assay Performance
331 Limit of Detection at 95 for One-Step and Two-StepMethods The results of LOD 95 for the ten kits tested usingtwo-step and one-step methods are recorded in Table 4
(i) Rotor Gene Q MDx The results showed an LOD 95 fortwo-step and one-step kits ranging from 41 to 171 copies120583Land from 20 to 497 copies120583L respectively (Table 4) Ofthe five commercial master mixes tested using the two-stepqPCR the RT SuperScript Vilo in combination with theExpress kit yielded greater sensitivity with an LOD 95 of41 copies120583L Maxima in combination with the RT Maximaand Perfecta in combination with the RT qScript rankedsecond and third with detection of 78 and 88 copies120583Lrespectively followed by the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 110 and 171 copies120583L respectively
In contrast the one-step qRT-PCR kit Quantitect-1 stepyielded the best levels of detection with an LOD 95 of20 copies120583L The four other tested one-step kits yieldedpoor detection with an LOD 95 varying between 127 and497 copies120583L
(ii) Mx3005P The LOD 95 for one-step and two-step kitsranged from 15 to 981 copies120583L and 45 to 160 copies120583L
respectivelyOf the five commercialmastermixes tested usingthe two-step qPCR the RT SuperScript Vilo in combinationwith the Express kit yielded greater sensitivity with an LOD95 of 45 copies120583L Perfecta in combination with qScriptRT and Maxima in combination with RT Maxima rankedsecond and third with a detection of 67 and 76 copies120583Lrespectively followed by the the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 88 and 160 copies120583L respectively
Of the five tested one-step kits two kits (ie Rotorgene-1 step and Quantitect-1 step) yielded the best levelsof detection with an LOD 95 of 15 and 22 copies120583Lrespectively SSIII Platinium-1 step ranked third with an LOD95 of 50 copies120583L followed by Verso-1 step (LOD 95 of101 copies120583L) and qScript-1 step (981 copies120583L)
332 Reproducibility of One-Step and Two-Step MethodsTable 5 shows the analysis results of reproducibility of one-step and two-step assays
(i) Rotor Gene Q MDx For all optimised one-step or two-step PCR assays the interassay CVs were lower than 10demonstrating good reproducibility for all tested kits [23]The interassay CVs for the two-step kits and the one-step kitsranged from 174 to 295 and 135 to 427 respectively whilethe intra-assay CVs ranged from 135 to 35 and 079 to 672for two-step and one-step kits respectively (Tables 5(a) and5(b)) In analysing the intra-assay CVs a somewhat similarvariability was shown for two-step and one-step reactions
The interassay CVs for the most efficient two-step kit(ie SuperScript Vilo in combination with qPCR Express)and the most efficient one-step kit (ie Quantitect-1 step)ranged from 195 to 336 respectively while the intra-assayCVs ranged from 185 to 211 and 079 to 216 for two-step andone-step kits respectively (Tables 5(a) and 5(b))
(ii) Mx3005P For all optimised one-step or two-step PCRassays the interassay CVs were lower than 10 demonstrat-ing good reproducibility for all tested kitsThe interassay CVsfor the two-step and one-step kits ranged from 214 to 33and 084 to 45 respectively while the intra-assay CVs rangedfrom 123 to 422 and 038 to 47 for two-step and one-stepkits respectively
34 Practicability of the Different Assays Costs and TimeTable 6 shows a practicability assessment of the differentreal-time assays including a comparison of cost pipettingsteps and thermocycling times Total reaction times for theten real-time assays ranged from 2h42 to 3h08 for two-stepkits and 2h18 to 2h57 for one-step kits with the one-stepSSIII Platinium kit requiring the shortest time and two-stepMaxima kit the longest regardless the machine used Thekits were ranked from 1 to 10 based on the cost per PCRreaction in increasing order from C108 (least expensive =1) to C1225 (most expensive = 10) Overall of the 10 mastermix kits tested the one-step kits were cheaper than the two-step kits (Table 6) For the one-step kits qScript was theleast costly followed by the Rotor Gene-1 step Verso-1 stepSSIII Platinium-1 step and the Quantitect-1 step which was
8 BioMed Research International
Table3Com
paris
onof
qRT-PC
RSY
BRGreen
(one-step)
kits
Results
offived
ifferentq
RT-PCR
kitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1in
vitro
RNAtranscrip
tsTestingof
different
qRT-PC
Rkits(one-step)
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Quantitect-1ste
pRo
torG
ene-1step
Verso-1step
SSIIIP
latinium-1ste
pqScript-1
step
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
2010
61666plusmn008
045
1715plusmn005
029
1699plusmn006
038
1452plusmn019
032
1347plusmn003
019
1779plusmn024
132
1588plusmn022
136
1834plusmn032
172
1622plusmn004
022
1848plusmn004
020
105
2010
52006plusmn010
052
2062plusmn004
017
2040plusmn011
052
1790plusmn021
118
1655plusmn003
019
2113plusmn045
212
1942plusmn020
104
2160plusmn015
071
1964plusmn041
211
2238plusmn015
068
104
2010
42358plusmn014
058
2391plusmn
008
033
2382plusmn020
083
2143plusmn012
057
1986plusmn004
018
2460plusmn020
083
2287plusmn016
069
2517plusmn017
069
2308plusmn027
116
2611plusmn010
038
103
2010
32670plusmn024
091
2745plusmn028
101
2693plusmn003
011
2490plusmn023
092
2313plusmn016
067
2795plusmn026
093
2634plusmn033
124
2863plusmn030
106
2614plusmn010
038
2954plusmn025
086
102
2010
23042plusmn008
026
23lowastlowast
3019plusmn060
198
03lowastlowast
03lowastlowast
3198plusmn017
053
2977plusmn039
132
3174plusmn079
250
2893plusmn008
028
3107plusmn037
118
101
2010
13324plusmn029
087
13lowastlowast
3327plusmn15
3460
03lowastlowast
03lowastlowast
3434plusmn10
3299
3284plusmn044
133
23lowastlowast
2991plusmn
010
033
03lowastlowast
12
03lowastlowast
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
03lowastlowast
03lowastlowast
Efficiency
98
96
100
94
104
97
94
95
100
87
1198772
0998
0999
0999
0998
0999
099
6099
8099
7099
6099
8
119910-in
tercept
3689
3767
3699
3529
3278
3815
3680
3893
3893
4074
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 9
Table4Detectio
nlim
itof
PCR(LOD95)for
two-ste
pandon
e-ste
pSY
BRGreen
RT-PCR
Two-ste
pRT
-qPC
RIScriptR
T+qP
CRSsoF
ast
BioR
adQuantiTectR
T+qP
CRQuantitect
QIAGEN
Maxim
aRT+qP
CRMaxim
aFE
RMEN
TAS
SSViloRT
+qP
CREx
press
INVITRO
GEN
qScriptR
T+qP
CRPerfe
cta
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
11055
171
8678
3941
2188
4488
44160
8076
3845
2267
34One-stepqR
T-PC
RQuantitect-1ste
pQIAGEN
RotorG
ene-1step
QIAGEN
Verso-1step
FERM
ENTA
SSSIIIP
latinium-1ste
pIN
VITRO
GEN
qScript-1
step
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
2040
497
994
127
254
321
641
406
812
2244
1530
101
202
50100
981
1962
lowastAllLO
D95results
werec
arrie
dou
tusin
gProb
itmod
ellowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
10 BioMed Research International
Table5Analysis
ofreprod
ucibilityof
one-ste
pandtwo-ste
pRT
-PCR
(a)
show
sfor
each
two-ste
pkittheintra-a
ndinterassay
reprod
ucibilityforsixdilutio
nsof
EBLV
-1teste
deighttim
esin
each
ofthreeind
ependent
runs
(119899=144samples)a
nd(b)sho
wsthe
inter-andintra-assayreprod
ucibilityfora
llteste
don
e-ste
pkits
(a)2-ste
pmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
IScriptR
T+qP
CRSsoF
ast
100
3046plusmn041
135
83008plusmn057
190
82982plusmn041
138
83012plusmn046
154
3012plusmn052
174
503111plusmn093
299
63184plusmn10
1319
63080plusmn046
151
83125plusmn080
256
3120plusmn088
283
253249plusmn059
183
63160plusmn067
212
83127plusmn068
219
63179plusmn065
204
3177plusmn080
251
103238plusmn091
281
63269plusmn119
363
63197plusmn076
239
53235plusmn095
294
3237plusmn097
299
53314
mdash1
mdashmdash
03198plusmn005
015
33256plusmn005
015
3227plusmn058
181
05
3217
mdash1
mdashmdash
03211plusmn076
236
33214plusmn076
236
3213plusmn062
193
250
3071plusmn
049
161
830
70plusmn039
128
830
61plusmn
033
108
830
67plusmn041
132
3067plusmn040
129
100
3230plusmn074
228
83203plusmn090
281
83219plusmn040
124
83217plusmn068
211
3217plusmn069
214
5034
02plusmn093
273
63324plusmn048
144
83316plusmn093
287
83347plusmn079
235
3342plusmn085
255
2534
63plusmn10
2295
83378plusmn075
223
634
24plusmn088
258
634
21plusmn
089
259
3425plusmn093
272
103595plusmn11
2312
53526plusmn047
134
534
68plusmn00
63
180
63530plusmn074
209
3526plusmn090
256
53384plusmn062
184
3mdash
mdash0
mdashmdash
034
83plusmn060
171
3384plusmn062
184
QuantiTectR
T+qP
CRQuantitect
250
3137plusmn069
220
83145plusmn052
165
83122plusmn046
147
83135plusmn056
177
3135plusmn055
175
100
3246plusmn080
246
73272plusmn092
281
73390plusmn090
265
73303plusmn087
264
3303plusmn10
5318
503359plusmn060
179
43434plusmn232
675
53366plusmn079
235
63386plusmn12
4363
3387plusmn14
0413
253298plusmn074
224
43358plusmn118
351
53412plusmn207
608
43356plusmn13
3394
3356plusmn13
8410
103354plusmn018
054
33386plusmn061
180
23473plusmn062
179
23404plusmn047
138
3397plusmn065
192
5mdash
mdash0
3552
mdash1
mdashmdash
03552
mdash3552
mdash50
034
09plusmn036
135
836
37plusmn118
326
63297plusmn038
116
834
30plusmn15
419
234
30plusmn15
4448
250
3642plusmn046
643
83366plusmn080
239
83361plusmn
083
246
834
56plusmn19
7376
3456plusmn19
7569
100
3439plusmn234
257
834
60plusmn098
284
834
50plusmn091
265
834
50plusmn089
269
3450plusmn089
258
5036
28plusmn088
475
63507plusmn091
259
734
92plusmn10
4297
73538plusmn13
2344
3538plusmn13
2374
253597plusmn17
2230
536
28plusmn088
242
436
44plusmn078
214
536
23plusmn079
229
3623plusmn079
217
10mdash
mdash0
mdashmdash
03765plusmn079
210
23765plusmn079
210
3765plusmn079
210
BioMed Research International 11
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
Maxim
aRT
+qP
CRMaxim
a
100
3183plusmn084
263
3138plusmn022
070
3009plusmn041
137
311plusmn049
157
311plusmn092
295
503335plusmn10
9326
83342plusmn088
264
53112plusmn064
205
83263plusmn087
265
3251plusmn
140
432
253367plusmn088
261
53277plusmn112
340
63247plusmn072
221
73297plusmn090
274
3290plusmn099
302
103716plusmn464
1249
33400plusmn14
1416
43435plusmn13
3388
53517plusmn246
684
3494plusmn263
754
53544plusmn024
068
23865
mdash1
3458plusmn12
4357
33622plusmn074
212
3555plusmn17
6496
25
mdashmdash
03314
mdash1
3347plusmn17
6526
23330plusmn17
6526
3336plusmn12
6377
250
2928plusmn058
199
829
05plusmn038
130
829
04plusmn039
135
829
12plusmn045
155
2912plusmn045
156
100
3053plusmn056
185
830
17plusmn048
159
830
31plusmn
043
143
830
34plusmn050
162
3034plusmn050
164
503154plusmn12
5397
83188plusmn13
7429
83172plusmn056
177
83171plusmn
108
334
3171plusmn
108
340
253300plusmn115
348
63238plusmn079
243
73238plusmn082
254
73256plusmn092
282
3256plusmn092
281
103308plusmn064
193
33302plusmn13
8417
43340plusmn14
6438
43317plusmn115
349
3317plusmn11
5347
503303plusmn015
045
23355
mdash1
3299
mdash1
3315plusmn028
045
3315plusmn028
085
qScriptR
T+qP
CRPerfe
cta
250
2704plusmn024
088
82675plusmn026
098
82677plusmn024
090
82685plusmn025
092
2685plusmn027
101
100
3054plusmn054
176
82984plusmn054
182
83046plusmn10
7350
83028plusmn072
236
3028plusmn079
261
503203plusmn290
905
73072plusmn089
292
83078plusmn082
267
83118plusmn15
4488
3114plusmn17
7568
253250plusmn098
302
73241plusmn
133
410
73363plusmn216
642
53285plusmn14
9452
3276plusmn14
9456
103222plusmn081
252
53287plusmn046
139
33270plusmn056
171
23260plusmn061
187
3251plusmn
069
211
53278plusmn015
045
2mdash
mdash0
3356plusmn15
5461
23318plusmn085
253
3318plusmn10
0303
250
2947plusmn022
074
829
49plusmn054
183
729
82plusmn038
129
829
60plusmn041
129
2960plusmn041
139
100
3099plusmn10
9352
83114plusmn12
6404
83102plusmn052
169
83105plusmn096
308
3105plusmn096
311
503187plusmn060
188
83137plusmn057
180
83313plusmn11
2337
83212plusmn10
8235
3212plusmn10
8335
253335plusmn299
896
73249plusmn068
211
83271plusmn
087
265
83283plusmn17
2457
3283plusmn17
2524
103300plusmn049
147
53345plusmn018
053
23298plusmn074
225
43307plusmn054
142
3307plusmn054
165
53304plusmn10
1306
23303plusmn074
223
23359
mdash1
3314plusmn067
265
3314plusmn067
203
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
2 BioMed Research International
the 1980s and due to the immense versatility and sensitivityof PCR diagnostic methods based on PCR technology havebeen extensively applied to many infectious diseases [5]To date real-time PCR is the state-of-the-art technologyfor the quantification of nucleic acids Various nucleic aciddetection-based assays have thus been published for thedetection of Lyssavirus fragments using conventional gel-based PCR NASBA or real-time PCR [6] In Europe mostlaboratories working on rabies employ conventional andorreal-time PCR [7] due to their high specificity and sensitivityThe conventional methods are reported to be very sensitivewhen using a heminested RT-PCR [8] and compared to hem-inested RT-PCR real-time PCR is shown to be about 100ndash1000-fold more sensitive [9] However due to this high PCRsensitivity which engenders a risk of sample cross contamina-tion and therefore a risk of potential false positive results RT-PCR is not recommended by the World Health Organizationfor routine postmortem diagnosis of rabies in animals [4]
Real-time RT-PCR which provides rapid sensitive andspecific detection of rabies virus decreases the risk of samplecontamination Various real-time assays have thus beendescribed since 2004 [10] for the rapid detection of RABVor other Lyssavirus species [11 12] using the one-step method[13 14] or the two-step method [12 15 16] Two systems ofreal-time assays are widely used for the detection of RABVand lyssaviruses the SYBR Green real-time RT-PCR andthe TaqMan real-time RT-PCR which is to date the mostcommon technique implemented in the laboratories workingon rabies [17]
As real-time PCR assays have become routine in diagnos-tic laboratories and because of their prominence there hasbeen rapid growth of commercially available reagent systemsand detection platforms which greatly increase the optionsavailable to laboratories In this study we evaluated severalcommercial master mixes in order to identify those whichproduce the best results using one-step and two-step real-time RT-PCR for the determination of EBLV-1 Five differentsystems for the generation of cDNA five two-step PCR kitsand five one-step qRT-PCR kits were comparedThe analysedperformance evaluation criteria included the generation ofa standard curve reaction efficiency analytical sensitivityintra- and interassay repeatability as well as the costs and thepracticability of kits and thermocycling times The goal ofthis study was also to determine if the one-step and two-stepsystems had the same reaction efficiencies for the detectionof EBLV-1 and if one system was more sensitive than anotherusing two real-time thermocyclers
2 Materials and Methods
21 Rabies Virus Isolates and RNA Extraction The Europeanbat Lyssavirus type 1 virus (EBLV-1) was selected for this studyfor the generation of in vitroRNA transcriptThe virus EBLV-1 (ANSES strain numbered 05-09) was isolated in France in2000 from a rabid bat (Eptesicus serotinus) The isolate wasmouse passaged before RNA extraction
Viral RNAwas extracted from 200120583L of 10mouse braintissue suspension using the Iprep Pure Link Virus kit (Invit-rogen France) according to the manufacturerrsquos instructions
The RNA concentration was determined spectrophotometri-cally and the RNA was stored at minus70∘C until use
22 Preparation of In Vitro RNA Transcripts Briefly a one-step RT-PCR amplification was performed as previouslydescribed [8] on 5 120583L of extracted RNA (ie 250ndash500 ng)with specific rabies primers (JW12 (forward) 51015840-ATGTAA-CACCYCTACAATG and JW6 (reverse) 51015840-CARTTVGCR-CACATYTTRTG) The 606 bp PCR products were purifiedand inserted into a pGEM T easy vector (Promega France)according to the manufacturerrsquos instructions Recombinantplasmids were purified using the PerfectPrep EndoFreePlasmid Maxi Kit (5Prime France) and then concentratedusing a step of sodium acetateethanol precipitation and lin-earised with the PstI restriction enzyme (Promega France)The clones of PCR products were bidirectionally sequencedwith sense and antisense primers T7 and SP6 by BeckmanCoulter Genomics (Takeley United Kingdom) to confirm thepresence of the target insert
In vitro transcription was performed using T7 RNApolymerase according to the manufacturerrsquos instructions(MaxiScript SP6T7 kit Ambion France)The resulting RNAtranscripts were purified as recommended by the manufac-turer using 2120583L of DNAse I during 15min at 37∘C followedby a final purification step using the RNeasymini kit (QiagenFrance) The RNA concentration was determined by usingthe Qubit Fluorometer (Invitrogen France) RNA transcriptswere aliquoted to 107 copies120583L and then stored at minus70∘CTheRNAmolecule copy numberwas calculated following themanufacturerrsquos instructions (MaxiScript SP6T7 kit AmbionFrance) using the following formula 119884 molecules120583L =(119883 g120583L RNA[transcript length in nucleotides times 330] times6022sdot1023) The estimated number of copies was of 268 sdot 1011for EBLV-1 RNA transcript
The quality of RNA transcript was checked on serialtenfold dilutions (106 to 1 copie120583L) through measurementsof efficiency of amplification using a two-step SYBR GreenRT-PCR [12] PCR efficiency was of 99
23 Real-Time RT-PCR Assays
231 Primers Used Universal pan Lyssavirus primers wereselected for this study Two questionnaires respectivelyundertaken in 2011-2012 by the EURL laboratory havedemonstrated that 13 out of 21 National Reference Laborato-ries for Rabies (NRLs) used primers JW12 and N165-146 pre-viously described by Wakeley et al [18] These specific rabiesprimers (JW12 (forward) ATGTAACACCYCTACAATGand N165-146 (reverse) GCAGGGTAYTTRTACTCATA)were designed to detect the rabies virus N gene of all knownlyssaviruses [12]
232 Two-Step RT-qPCR Assays
(1) Commercial Master Mix Kits
(a) Reverse Transcription Kits (Table 1(a)) Five differentreverse transcription (RT) systems provided by five differentcompanies and five different qPCR kits provided by the same
BioMed Research International 3
Table 1 Characteristics of the commercial master mix kits used In (a) the reactions mix and thermocycling conditions for the five RT kitsare detailed in (b) the reactions mix and thermocycling conditions for the five qPCR SybrGreen kits (two-step) and in (c) the reactions Mixand thermocycling conditions for the five qRT-PCR SybrGreen kits (one-step) used in this study
(a) Reactions mix and thermocycling conditions for the five RT kits
Manufacturer Reversetranscription kit RTase and primers Thermocycling
conditions Reaction mixlowastlowast
Bio-Rad Iscript select cDNAsynthesis kit
M-MLV RTlowastRandom primer
5min-25∘C30min-42∘C5min-85∘C
4 120583L Iscript Mix (5x) + 2120583L Random primer +1 120583L Iscript RTase + 8 120583L RNase free water
QiagenQuantiTectReverse
Transcription Kit
Quantiscript RTaseMix OligodT
+ random primer
2min-42∘C30min-42∘C3min-95∘C
2120583L gDNAWipeout Buffer (7x) + 4 120583LQuantiscript RT Buffer + 1120583L QuantiscriptRTase + 1 120583L RT primer + 7 120583L RNase free
water
FermentasMaxima HMinusFirst Strand cDNA
Synthesis Kit
M-MLV RTlowastRandom primer
5min-65∘C10min-25∘C30min-50∘C5min-85∘C
4120583L RT Buffer (5x) + 1 120583L Random primer +1 120583L dNTP (10mM) + 1 120583L Maxima HMinus
Ez Mix + 8 120583L RNase free water
Invitrogen SuperScript ViloMaster Mix
M-MLV RTlowastRandom primer
10min-25∘C30min-42∘C5min-85∘C
4120583L SuperScript Vilo MasterMix (5x) + 11 120583LRNase free water
Quanta qScript cDNASynthesis Kit
M-MLV RTlowastMix OligodT
+ random primer
5min-22∘C30min-42∘C5min-85∘C
4 120583L qScript Reaction Mix (5x) + 1 120583L qScriptRTase + 10 120583L RNase free water
lowastMoloney Murine Leukemia ViruslowastlowastTotal volume of reaction is 20120583L containing 5120583L of RNA sample and 15 120583L of reaction mix All RT assays were performed on the Mastercycler Eppendorfthermal cyclerRTase reverse transcriptase
(b) Reactions mix and thermocycling conditions for the five qPCR SybrGreen kits (two-step)
Manufacturer qPCRSybrGreen kit Thermocycling conditions Reaction mixlowast
Bio-RadSsoFastEvaGreenSupermix
30 sec-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 20 120583L containing 2 120583L of cDNA and18 120583L of a reaction mix (1x Sso Fast master mix035 120583M [05 120583M] each of pan-Lyssavirus primers)
QiagenQuantiTectSYBR GreenPCR Kit
15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 25 120583L containing 2 120583L of cDNA and23 120583L of reaction mix (1x Quantitect Master mix05 120583M [06 120583M] each of pan-Lyssavirus primers)
FermentasMaxima SYBRGreen qPCRMaster Mix
10min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 25 120583L containing 2 120583L of cDNA and23 120583L of reaction mix (1x Maxima SYBR Green mastermix 04 120583M [1 120583M] each of pan-Lyssavirus primers)
Invitrogen
Express SYBRGreenER qPCR
SupermixUniversal
2min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 20 120583L containing 2 120583L of cDNA and18 120583L of reaction mix (1x Express SYBR GreenERSupermix 04 120583M [2 120583M] each of pan-Lyssavirus
primers) [50 nM of ROX dye]
Quanta Perfecta SYBRGreen SuperMix
3min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 25 120583L containing 2 120583L of cDNA and23120583L of reaction mix (1x Perfecta SYBR GreenSupermix 03 120583M [1 120583M] each of pan-Lyssavirus
primers)lowastThe PCR reaction protocol corresponds to PCR assays performed on the Rotor Gene Q MDx The concentrations of primers used on the real timethermocycler Mx3005P are shown in brackets [ ]
(c) Reactions mix and thermocycling conditions for the five qRT-PCR SYBR Green kits (one-step)
Manufacturer qRT-PCRSybrGreen kit Thermocycling conditions Reaction mixlowast
Qiagen QuantiTect SYBRGreen RT-PCR Kit
30min-50∘C 15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x QuantiTect SYBR Green master mix1 Quantitect RT mix 06 120583M [2120583M]
each of pan-Lyssavirus primers
4 BioMed Research International
(c) Continued
Manufacturer qRT-PCRSybrGreen kit Thermocycling conditions Reaction mixlowast
QiagenRotor Gene-1 stepRotor-Gene SYBRGreen RT-PCR Kit
30min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x Rotor-Gene master mix 1Rotor-Gene RT mix 1 120583M [2 120583M] each of
pan-Lyssavirus primers
Fermentas Verso 1-stepQRT-PCR Kit
30min-50∘C 15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x Verso SYBR Green master mix 5 RTenhancer 1 Verso enzyme mix 02 120583M[03 120583M] each of pan-Lyssavirus primers
Invitrogen
SuperScript IIIPlatinum SYBRGreen One-StepqRT-PCR Kit
10min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x SuperScript III Platinium master mix2 enzyme mix 03 120583M [05120583M] each of
pan-Lyssavirus primers
QuantaqScript One-StepSYBR GreenRT-qPCR Kit
30min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x qScript master mix 2 qScript enzymemix 04 120583M [02 120583M] each of
pan-Lyssavirus primerslowastTotal volume of reaction is 25 120583L containing 2120583L of RNA sample and 23120583L of reactionmixThe PCR reaction protocol corresponds to PCR assays performedon the Rotor Gene Q MDx The concentrations of primers used on the real time thermocycler Mx3005P are shown in brackets [ ]
five suppliers were tested with the two-step method Thefollowing RT kits were compared Iscript (IScript cDNA syn-thesis kit Bio-Rad France) QuantiTect (Quantitect Reversetranscription kit Qiagen France) Maxima (Maxima HMinus First Strand cDNA synthesis kit Fermentas France)SS Vilo (Superscript Vilo Master Mix Invitrogen France)and qScript (qScript cDNA Synthesis kit Quanta France)
(b) Two-Step SYBR Green PCR Kits (Table 1(b)) Differenttwo-step qPCR kits were tested SsoFast (SsoFast EvagreenSuper mix kit Bio-Rad France) QuantiTect (QuantiTectSYBR Green PCR kit Qiagen France) Maxima (MaximaSYBR Green qPCR master mix Fermentas France) Express(Express SYBR GreenER qPCR Supermix universal Invitro-gen France) and Perfecta (Perfecta SYBR Green SupermixQuanta France)
(2) Two-Step RT-qPCR Analysis Seven 1 10 serial dilutions(ie 106 to 1 copie120583L) of the in vitro EBLV-1 RNA transcriptwere prepared in RNAse free water then subjected to reversetranscription using the five different RT master mixes AllRT kits were optimised with regard to the time and the tem-perature of incubation (Table 1(a)) Complementary cDNAswere generated from 5120583L of each dilution of EBLV-1 RNArandomprimer oligo dT or amix of oligo dTrandomprimerincluded in each tested kit according to the recommendationsof themanufacturersThe cycling conditions and the reactionprotocol for each tested RT kit are detailed in Table 1(a)Ther-mocycling was performed on the Mastercycler Eppendorfthermal cycler (Eppendorf France)
Real-time PCRs were run on the thermocyclers RotorGene QMDx (Qiagen France) and Mx3005P (Agilent Tech-nologies France) The concentrations of the reverse (N165-146) and forward (JW12) primers were optimised for eachtested kit At least three different concentrations were exam-ined that is the one recommended by the manufacturerone below and one above the recommended primer concen-tration using the qPCR conditions described in Table 1(b)The optimised concentration of primers is recorded for each
tested qPCR kits in Table 1(b) Reactions were performed intriplicate in a total volume of 20 120583L or 25 120583L following themanufacturerrsquos instructions (Table 1(b))
The following thermal profile was used PCR initialactivation step at 95∘C for 30 sec (SsoFast) 15min (Quan-tiTect) 10min (Maxima) 2min (Express) or 3min (Per-fecta) according to themanufacturerrsquos recommendationsThecycler conditions performed as previously described byHay-man et al [19] were the same for all assays 45 cycles of three-step cycling consisting of denaturation at 94∘C for 30 secannealing at 55∘C for 30 sec and extension at 72∘C for 30 sec
233 One-Step qRT-PCR Assays
(1) Commercial qRT-PCR Kits (Table 1(c)) Five commer-cial one-step kits provided by four suppliers were testedQuantiTect-1 step (QuantiTect SYBR Green RT-PCR kitQiagen France) RotorGene-1 step (Rotor-Gene SYBRGreenRT-PCR kit Qiagen France) Verso-1 step (Verso SYBRGreen one-step qRT-PCR kit Fermentas France) SSIIIPlatinium-1 step (SuperScript III Platinium SYBR GreenOne-step qRT-PCR kit Invitrogen France) and qScript-1step (qScript One step SYBR Green QRT-PCR kit QuantaFrance)
(2) One-Step qRT-PCR Analysis To compare the five mastermixes the assays were performed in triplicate using seventenfold dilutions (ie 106 to 1 copies120583L) of EBLV-1 RNAPCR was performed using a total volume of 25 120583L containing2 120583L of serial dilutions of EBLV-1 RNA and the master mixcomponents (119881 = 23 120583L) from each representative kit whichwere prepared according to the respective manufacturerrsquosinstructions (Table 1(c)) The concentrations of the reverse(N165-146) and forward (JW12) primers were optimised foreach tested kit (Table 1(c)) At least three different con-centrations were examined the one recommended by themanufacturer one below and one above the recommendedprimer concentration (Table 1(b))
BioMed Research International 5
Real-time RT-PCR assays were performed on the ther-mocycler Rotor Gene Q MDx and Mx3005P using the samethermocycling conditions for the five kits (ie 45 cycles of30 sec-94∘C 30 sec-55∘C and 30 sec-72∘C) For each testedkit the DNA polymerase activation step was performed asrecommended by the supplier (Table 1(c))
234 Data Collection and Analysis PCR efficiency and coef-ficient of determination (1198772) values were calculated by thesoftware Rotor Gene Q Series and Mx3005Pro respectively
Negative and positive controls were included in eachassay in which a threshold setting (Ct) of 003 was used as thereference for the Rotor Gene QMDx thermocycler A thresh-old setting of 300 was used as the reference for the Mx3005Pthermocycler for all tested kits except for one two-step kit(Express) for which a threshold setting of 005 was used
24 Determination of the Performance of the Method Theperformance of the method was determined for all testedkits following OIE recommendations [20] and those of theFrench Agency for Standard Operating Procedure for PCRexperimental design [21] Performance testingwas conductedfor the two real-time platforms
241 Determination of PCR Efficiency A 106 to 103 copies120583Ldilution range corresponds to the linear dynamic range forthe ten tested kits To determine the PCR efficiency foreach tested kit a standard curve was plotted based on Ctvalues obtained from samples containing 106 to 103 copies120583Lagainst the log of the estimated RNA copy number in thesample The coefficient of determination (1198772) was calculatedand considered as suitable when not lower than 099 Theslope of the standard curves was used for the determinationof PCR efficiency (119864 = 10[minus1slope] minus 1) The efficiency wasconsidered satisfactory when it is not lower than 90 and notabove 110 [22]
242 Determination of the Limit of Detection at 95 Thelimit of detection at 95 (LOD 95) which corresponds tothe lowest RNA concentration that could be detected with95 confidence was calculated using the inverse standardnormal distribution calculation [21] The LOD 95 wascalculated using the Probit analysis based on the results ofthree runs of eight repetitions of six dilutions each of EBLV-1RNA in RNase-free water (119899 = 144 samples tested) The sixdilutions (cDNA and RNA) were selected surrounding thelimit of detection of real-time PCR assays
On the Rotor Gene Q MDx gene cycler 103 to 25 cop-iessample were tested with the kit Rotor Gene-1 step 103to 50 copiessample for qScript-1 stepSSIII Platinium-1 step500 to 10 copiessample for Verso-1 step 50 to 1 copiessamplefor Quantitect-1 step 250 to 5 copiessample for Perfecta 250to 5 copiessample for QuantiTect 102 to 25 copiessamplefor Maxima 100 to 05 copiessample for Sso Fast and 100 to05 copiessample for Express
On Mx3005P cycler 2500 to 100 copiessample weretested with the kit qScript-1 step 250 to 5 copiessample forSSIII Platinium-1 stepVerso-1 step 50 to 1 copiessample forRotor Gene-1 stepQuantitect-1 step 500 to 10 copiessample
for QuantiTect 250 to 5 copies120583L for MaximaSso FastPer-fecta 250 to 5 copiessample for Perfecta and 100 to 25 cop-iessample for Express
Three independent assays were undertaken under identi-cal laboratory conditions
243 Evaluation of Reproducibility To evaluate the repro-ducibility of the ten kits inter- and intraspecific assay repro-ducibility were assessed using the coefficient of variation(CV) of theCt values for the lowest RNAconcentration show-ing 100 positive results The intra-assay repeatability wasdetermined using three independent runs of eight replicatesof EBLV-1 (119899 = 24 samples tested) The interassay repeata-bility was assessed for the same serial replicates in threeindependent assays using identical laboratory conditionsCVs expressed as percentages were obtained by dividing thestandard deviation of each tested sample by the mean of thecycle threshold values and multiplying that result by 100
3 Results
31 Differences in RNADetection Levels Using Two-StepAssays
311 Reverse Transcription Kits Of the five reverse transcrip-tion kits tested in combination with the SSoFast kit for cDNAamplification the SuperScript Vilo yielded greater sensitivityand resulted in a detection limit of 10 copies120583L (3 positivesamples out of 3 tested) The efficiency of amplification wasof 98The four other RT kits (ie kits Quantitect MaximaSS Vilo and qScript) resulted in a detection of 100 copies120583LThe amplification efficiency varied between 98 and 104The 119910-intercept for all reverse transcription kits tested incombination with the qPCR SsoFast kit varied between 3513and 3746
312 Two-Step PCR Kits All optimised two-step qPCRassays tested in combination with the Iscript RT showed sim-ilar sensitivity and enabled detection of at least 100 copies120583Lof EBLV-1 on the Rotor Gene Q MDx gene cycler Theefficiency of amplificationwhichwas satisfactory for all testedkits varied between 90 (ie kit Perfecta) and 99 (ie kitsSSo Fast Quantitect SG Maxima SG and Express) with an119910-intercept varying between 3603 and 3857
313 Comparison of Two-Step RT-qPCR Kits Provided bythe Same Manufacturer Table 2 details the results of thecomparison of the five two-step kits
(i) Rotor Gene Q MDx Of the five PCR kits tested incombination with the reverse transcriptases provided by thesame manufacturer one RT-qPCR assay (SuperScript Vilo incombination with qPCR Express kit) yielded greater sensitiv-ity and showed a detection of at least 10 copies120583L (Table 2)All amplicons investigated with the two-step method usingqPCR kits and RT kits from the same provider showedsatisfactory efficiency of amplification (100 plusmn 10) The 119910-intercept for all tested kits varied between 3473 and 3955
(ii) Mx3005P Of the five tested RT-qPCR kits one RT-qPCRassay (Maxima RTqPCRMaxima) yielded greater sensitivity
6 BioMed Research International
Table2Com
paris
onof
RT-qPC
RSybrGreen
kits(tw
o-ste
p)provided
bythes
amem
anufacturerRe
sults
offived
ifferentq
PCRkitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1RN
A
Testingof
different
RT-qPC
Rkits(tw
o-ste
p)provided
bythes
amem
anufacturerlowastlowastlowastlowast
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
IScriptR
T+qP
CRSsoF
ast
QuantiTectR
T+qP
CRQuantitect
Maxim
aRT+qP
CRMaxim
aSS
ViloRT
+qP
CREx
press
qScriptR
T+qP
CRPerfe
cta
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
5010
51658plusmn013
077
1947plusmn007
034
1650plusmn015
092
1501plusmn
005
035
1609plusmn003
019
1896plusmn004
021
2279plusmn026
115
1747plusmn009
052
1749plusmn010
054
1799plusmn029
159
105
5010
41993plusmn009
045
2289plusmn010
042
1950plusmn006
033
1821plusmn
005
026
1979plusmn003
013
2217plusmn006
025
2594plusmn009
035
2116plusmn029
138
2095plusmn003
015
2127plusmn015
072
104
5010
32328plusmn005
020
2589plusmn006
025
2295plusmn028
123
2168plusmn017
076
2318plusmn016
067
2575plusmn005
018
2960plusmn042
143
2429plusmn029
039
2440plusmn012
049
2455plusmn008
033
103
5010
22661plusmn
021
080
2964plusmn032
107
2643plusmn026
098
2481plusmn
005
019
2694plusmn008
030
2916plusmn023
080
3210plusmn019
060
2778plusmn030
109
2803plusmn074
263
2784plusmn012
042
102
5010
13045plusmn013
042
3220plusmn041
126
3059plusmn045
146
2833plusmn064
225
3140plusmn053
168
3238plusmn061
189
3512plusmn010
028
3046plusmn092
303
3105plusmn038
123
3135plusmn070
222
101
5010
023lowastlowast
13lowastlowast
23lowastlowast
3152plusmn13
3420
13lowastlowast
13lowastlowast
13lowastlowast
3276plusmn070
212
13lowastlowast
23lowastlowast
105
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
Efficiency
99
99
100
101
90
96
107
97
93
102
1198772
0999
0997
0997
0999
0999
099
9099
1099
7099
4099
8
119910-in
tercept
3665
3955
3631
3473
3767
3938
4183
3801
3850
3801
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883
num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 7
and showed a detection of at least 10 copies120583L All RT-qPCRassays showed satisfactory efficiency varying between 93 and107 with detection of at least 100 copies120583LThe 119910-interceptvaried between 3801 and 4183 for all tested kits
32 Differences in RNA Detection between the Five Kits TestedUsing the One-Step Method Table 3 details the results of thecomparison of the five one-step kits
(i) Rotor Gene Q MDx Three optimised qRT-PCR assaysrevealed poor sensitivity and enabled detection of at least103 copies120583L Of the five kits tested two kits (QuantiTect-1 step and Verso-1 step) yielded greater sensitivity with thedetection of at least 10 copies120583L (3 positive samples out of3 tested) All kits showed a satisfactory efficiency varyingbetween 94 and 104The119910-intercept varied between 3278and 3767 for the five tested one-step kits
A high presence of Primer-Dimer was shown for lowRNA concentrations for three kits SSIII Platinium-1 stepVerso-1 step and qScript-1 step
(ii) Mx3005P Of the five one-step kits tested using theMx3005P cycler three optimised one-step kits (ie Quanti-tect-1 step Rotor Gene-1 step and SSIII Platinium-1 step)revealed good sensitivity with the detection of at least10 copies120583L The two other optimised one-step assays (ieVerso-1 step and qScript-1 step) revealed poor sensitivitywith a detection of 102 and 103 copies120583L respectively Theamplification efficiencywas satisfactory for all kits (94ndash100)except for the qScript one-step kit (efficiency of 87) The 119910-intercept varied between 3680 and 4074 for all tested kitsexcept for the qScript kit (Intercept of 4074)
33 Comparison of Assay Performance
331 Limit of Detection at 95 for One-Step and Two-StepMethods The results of LOD 95 for the ten kits tested usingtwo-step and one-step methods are recorded in Table 4
(i) Rotor Gene Q MDx The results showed an LOD 95 fortwo-step and one-step kits ranging from 41 to 171 copies120583Land from 20 to 497 copies120583L respectively (Table 4) Ofthe five commercial master mixes tested using the two-stepqPCR the RT SuperScript Vilo in combination with theExpress kit yielded greater sensitivity with an LOD 95 of41 copies120583L Maxima in combination with the RT Maximaand Perfecta in combination with the RT qScript rankedsecond and third with detection of 78 and 88 copies120583Lrespectively followed by the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 110 and 171 copies120583L respectively
In contrast the one-step qRT-PCR kit Quantitect-1 stepyielded the best levels of detection with an LOD 95 of20 copies120583L The four other tested one-step kits yieldedpoor detection with an LOD 95 varying between 127 and497 copies120583L
(ii) Mx3005P The LOD 95 for one-step and two-step kitsranged from 15 to 981 copies120583L and 45 to 160 copies120583L
respectivelyOf the five commercialmastermixes tested usingthe two-step qPCR the RT SuperScript Vilo in combinationwith the Express kit yielded greater sensitivity with an LOD95 of 45 copies120583L Perfecta in combination with qScriptRT and Maxima in combination with RT Maxima rankedsecond and third with a detection of 67 and 76 copies120583Lrespectively followed by the the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 88 and 160 copies120583L respectively
Of the five tested one-step kits two kits (ie Rotorgene-1 step and Quantitect-1 step) yielded the best levelsof detection with an LOD 95 of 15 and 22 copies120583Lrespectively SSIII Platinium-1 step ranked third with an LOD95 of 50 copies120583L followed by Verso-1 step (LOD 95 of101 copies120583L) and qScript-1 step (981 copies120583L)
332 Reproducibility of One-Step and Two-Step MethodsTable 5 shows the analysis results of reproducibility of one-step and two-step assays
(i) Rotor Gene Q MDx For all optimised one-step or two-step PCR assays the interassay CVs were lower than 10demonstrating good reproducibility for all tested kits [23]The interassay CVs for the two-step kits and the one-step kitsranged from 174 to 295 and 135 to 427 respectively whilethe intra-assay CVs ranged from 135 to 35 and 079 to 672for two-step and one-step kits respectively (Tables 5(a) and5(b)) In analysing the intra-assay CVs a somewhat similarvariability was shown for two-step and one-step reactions
The interassay CVs for the most efficient two-step kit(ie SuperScript Vilo in combination with qPCR Express)and the most efficient one-step kit (ie Quantitect-1 step)ranged from 195 to 336 respectively while the intra-assayCVs ranged from 185 to 211 and 079 to 216 for two-step andone-step kits respectively (Tables 5(a) and 5(b))
(ii) Mx3005P For all optimised one-step or two-step PCRassays the interassay CVs were lower than 10 demonstrat-ing good reproducibility for all tested kitsThe interassay CVsfor the two-step and one-step kits ranged from 214 to 33and 084 to 45 respectively while the intra-assay CVs rangedfrom 123 to 422 and 038 to 47 for two-step and one-stepkits respectively
34 Practicability of the Different Assays Costs and TimeTable 6 shows a practicability assessment of the differentreal-time assays including a comparison of cost pipettingsteps and thermocycling times Total reaction times for theten real-time assays ranged from 2h42 to 3h08 for two-stepkits and 2h18 to 2h57 for one-step kits with the one-stepSSIII Platinium kit requiring the shortest time and two-stepMaxima kit the longest regardless the machine used Thekits were ranked from 1 to 10 based on the cost per PCRreaction in increasing order from C108 (least expensive =1) to C1225 (most expensive = 10) Overall of the 10 mastermix kits tested the one-step kits were cheaper than the two-step kits (Table 6) For the one-step kits qScript was theleast costly followed by the Rotor Gene-1 step Verso-1 stepSSIII Platinium-1 step and the Quantitect-1 step which was
8 BioMed Research International
Table3Com
paris
onof
qRT-PC
RSY
BRGreen
(one-step)
kits
Results
offived
ifferentq
RT-PCR
kitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1in
vitro
RNAtranscrip
tsTestingof
different
qRT-PC
Rkits(one-step)
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Quantitect-1ste
pRo
torG
ene-1step
Verso-1step
SSIIIP
latinium-1ste
pqScript-1
step
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
2010
61666plusmn008
045
1715plusmn005
029
1699plusmn006
038
1452plusmn019
032
1347plusmn003
019
1779plusmn024
132
1588plusmn022
136
1834plusmn032
172
1622plusmn004
022
1848plusmn004
020
105
2010
52006plusmn010
052
2062plusmn004
017
2040plusmn011
052
1790plusmn021
118
1655plusmn003
019
2113plusmn045
212
1942plusmn020
104
2160plusmn015
071
1964plusmn041
211
2238plusmn015
068
104
2010
42358plusmn014
058
2391plusmn
008
033
2382plusmn020
083
2143plusmn012
057
1986plusmn004
018
2460plusmn020
083
2287plusmn016
069
2517plusmn017
069
2308plusmn027
116
2611plusmn010
038
103
2010
32670plusmn024
091
2745plusmn028
101
2693plusmn003
011
2490plusmn023
092
2313plusmn016
067
2795plusmn026
093
2634plusmn033
124
2863plusmn030
106
2614plusmn010
038
2954plusmn025
086
102
2010
23042plusmn008
026
23lowastlowast
3019plusmn060
198
03lowastlowast
03lowastlowast
3198plusmn017
053
2977plusmn039
132
3174plusmn079
250
2893plusmn008
028
3107plusmn037
118
101
2010
13324plusmn029
087
13lowastlowast
3327plusmn15
3460
03lowastlowast
03lowastlowast
3434plusmn10
3299
3284plusmn044
133
23lowastlowast
2991plusmn
010
033
03lowastlowast
12
03lowastlowast
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
03lowastlowast
03lowastlowast
Efficiency
98
96
100
94
104
97
94
95
100
87
1198772
0998
0999
0999
0998
0999
099
6099
8099
7099
6099
8
119910-in
tercept
3689
3767
3699
3529
3278
3815
3680
3893
3893
4074
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 9
Table4Detectio
nlim
itof
PCR(LOD95)for
two-ste
pandon
e-ste
pSY
BRGreen
RT-PCR
Two-ste
pRT
-qPC
RIScriptR
T+qP
CRSsoF
ast
BioR
adQuantiTectR
T+qP
CRQuantitect
QIAGEN
Maxim
aRT+qP
CRMaxim
aFE
RMEN
TAS
SSViloRT
+qP
CREx
press
INVITRO
GEN
qScriptR
T+qP
CRPerfe
cta
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
11055
171
8678
3941
2188
4488
44160
8076
3845
2267
34One-stepqR
T-PC
RQuantitect-1ste
pQIAGEN
RotorG
ene-1step
QIAGEN
Verso-1step
FERM
ENTA
SSSIIIP
latinium-1ste
pIN
VITRO
GEN
qScript-1
step
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
2040
497
994
127
254
321
641
406
812
2244
1530
101
202
50100
981
1962
lowastAllLO
D95results
werec
arrie
dou
tusin
gProb
itmod
ellowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
10 BioMed Research International
Table5Analysis
ofreprod
ucibilityof
one-ste
pandtwo-ste
pRT
-PCR
(a)
show
sfor
each
two-ste
pkittheintra-a
ndinterassay
reprod
ucibilityforsixdilutio
nsof
EBLV
-1teste
deighttim
esin
each
ofthreeind
ependent
runs
(119899=144samples)a
nd(b)sho
wsthe
inter-andintra-assayreprod
ucibilityfora
llteste
don
e-ste
pkits
(a)2-ste
pmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
IScriptR
T+qP
CRSsoF
ast
100
3046plusmn041
135
83008plusmn057
190
82982plusmn041
138
83012plusmn046
154
3012plusmn052
174
503111plusmn093
299
63184plusmn10
1319
63080plusmn046
151
83125plusmn080
256
3120plusmn088
283
253249plusmn059
183
63160plusmn067
212
83127plusmn068
219
63179plusmn065
204
3177plusmn080
251
103238plusmn091
281
63269plusmn119
363
63197plusmn076
239
53235plusmn095
294
3237plusmn097
299
53314
mdash1
mdashmdash
03198plusmn005
015
33256plusmn005
015
3227plusmn058
181
05
3217
mdash1
mdashmdash
03211plusmn076
236
33214plusmn076
236
3213plusmn062
193
250
3071plusmn
049
161
830
70plusmn039
128
830
61plusmn
033
108
830
67plusmn041
132
3067plusmn040
129
100
3230plusmn074
228
83203plusmn090
281
83219plusmn040
124
83217plusmn068
211
3217plusmn069
214
5034
02plusmn093
273
63324plusmn048
144
83316plusmn093
287
83347plusmn079
235
3342plusmn085
255
2534
63plusmn10
2295
83378plusmn075
223
634
24plusmn088
258
634
21plusmn
089
259
3425plusmn093
272
103595plusmn11
2312
53526plusmn047
134
534
68plusmn00
63
180
63530plusmn074
209
3526plusmn090
256
53384plusmn062
184
3mdash
mdash0
mdashmdash
034
83plusmn060
171
3384plusmn062
184
QuantiTectR
T+qP
CRQuantitect
250
3137plusmn069
220
83145plusmn052
165
83122plusmn046
147
83135plusmn056
177
3135plusmn055
175
100
3246plusmn080
246
73272plusmn092
281
73390plusmn090
265
73303plusmn087
264
3303plusmn10
5318
503359plusmn060
179
43434plusmn232
675
53366plusmn079
235
63386plusmn12
4363
3387plusmn14
0413
253298plusmn074
224
43358plusmn118
351
53412plusmn207
608
43356plusmn13
3394
3356plusmn13
8410
103354plusmn018
054
33386plusmn061
180
23473plusmn062
179
23404plusmn047
138
3397plusmn065
192
5mdash
mdash0
3552
mdash1
mdashmdash
03552
mdash3552
mdash50
034
09plusmn036
135
836
37plusmn118
326
63297plusmn038
116
834
30plusmn15
419
234
30plusmn15
4448
250
3642plusmn046
643
83366plusmn080
239
83361plusmn
083
246
834
56plusmn19
7376
3456plusmn19
7569
100
3439plusmn234
257
834
60plusmn098
284
834
50plusmn091
265
834
50plusmn089
269
3450plusmn089
258
5036
28plusmn088
475
63507plusmn091
259
734
92plusmn10
4297
73538plusmn13
2344
3538plusmn13
2374
253597plusmn17
2230
536
28plusmn088
242
436
44plusmn078
214
536
23plusmn079
229
3623plusmn079
217
10mdash
mdash0
mdashmdash
03765plusmn079
210
23765plusmn079
210
3765plusmn079
210
BioMed Research International 11
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
Maxim
aRT
+qP
CRMaxim
a
100
3183plusmn084
263
3138plusmn022
070
3009plusmn041
137
311plusmn049
157
311plusmn092
295
503335plusmn10
9326
83342plusmn088
264
53112plusmn064
205
83263plusmn087
265
3251plusmn
140
432
253367plusmn088
261
53277plusmn112
340
63247plusmn072
221
73297plusmn090
274
3290plusmn099
302
103716plusmn464
1249
33400plusmn14
1416
43435plusmn13
3388
53517plusmn246
684
3494plusmn263
754
53544plusmn024
068
23865
mdash1
3458plusmn12
4357
33622plusmn074
212
3555plusmn17
6496
25
mdashmdash
03314
mdash1
3347plusmn17
6526
23330plusmn17
6526
3336plusmn12
6377
250
2928plusmn058
199
829
05plusmn038
130
829
04plusmn039
135
829
12plusmn045
155
2912plusmn045
156
100
3053plusmn056
185
830
17plusmn048
159
830
31plusmn
043
143
830
34plusmn050
162
3034plusmn050
164
503154plusmn12
5397
83188plusmn13
7429
83172plusmn056
177
83171plusmn
108
334
3171plusmn
108
340
253300plusmn115
348
63238plusmn079
243
73238plusmn082
254
73256plusmn092
282
3256plusmn092
281
103308plusmn064
193
33302plusmn13
8417
43340plusmn14
6438
43317plusmn115
349
3317plusmn11
5347
503303plusmn015
045
23355
mdash1
3299
mdash1
3315plusmn028
045
3315plusmn028
085
qScriptR
T+qP
CRPerfe
cta
250
2704plusmn024
088
82675plusmn026
098
82677plusmn024
090
82685plusmn025
092
2685plusmn027
101
100
3054plusmn054
176
82984plusmn054
182
83046plusmn10
7350
83028plusmn072
236
3028plusmn079
261
503203plusmn290
905
73072plusmn089
292
83078plusmn082
267
83118plusmn15
4488
3114plusmn17
7568
253250plusmn098
302
73241plusmn
133
410
73363plusmn216
642
53285plusmn14
9452
3276plusmn14
9456
103222plusmn081
252
53287plusmn046
139
33270plusmn056
171
23260plusmn061
187
3251plusmn
069
211
53278plusmn015
045
2mdash
mdash0
3356plusmn15
5461
23318plusmn085
253
3318plusmn10
0303
250
2947plusmn022
074
829
49plusmn054
183
729
82plusmn038
129
829
60plusmn041
129
2960plusmn041
139
100
3099plusmn10
9352
83114plusmn12
6404
83102plusmn052
169
83105plusmn096
308
3105plusmn096
311
503187plusmn060
188
83137plusmn057
180
83313plusmn11
2337
83212plusmn10
8235
3212plusmn10
8335
253335plusmn299
896
73249plusmn068
211
83271plusmn
087
265
83283plusmn17
2457
3283plusmn17
2524
103300plusmn049
147
53345plusmn018
053
23298plusmn074
225
43307plusmn054
142
3307plusmn054
165
53304plusmn10
1306
23303plusmn074
223
23359
mdash1
3314plusmn067
265
3314plusmn067
203
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 3
Table 1 Characteristics of the commercial master mix kits used In (a) the reactions mix and thermocycling conditions for the five RT kitsare detailed in (b) the reactions mix and thermocycling conditions for the five qPCR SybrGreen kits (two-step) and in (c) the reactions Mixand thermocycling conditions for the five qRT-PCR SybrGreen kits (one-step) used in this study
(a) Reactions mix and thermocycling conditions for the five RT kits
Manufacturer Reversetranscription kit RTase and primers Thermocycling
conditions Reaction mixlowastlowast
Bio-Rad Iscript select cDNAsynthesis kit
M-MLV RTlowastRandom primer
5min-25∘C30min-42∘C5min-85∘C
4 120583L Iscript Mix (5x) + 2120583L Random primer +1 120583L Iscript RTase + 8 120583L RNase free water
QiagenQuantiTectReverse
Transcription Kit
Quantiscript RTaseMix OligodT
+ random primer
2min-42∘C30min-42∘C3min-95∘C
2120583L gDNAWipeout Buffer (7x) + 4 120583LQuantiscript RT Buffer + 1120583L QuantiscriptRTase + 1 120583L RT primer + 7 120583L RNase free
water
FermentasMaxima HMinusFirst Strand cDNA
Synthesis Kit
M-MLV RTlowastRandom primer
5min-65∘C10min-25∘C30min-50∘C5min-85∘C
4120583L RT Buffer (5x) + 1 120583L Random primer +1 120583L dNTP (10mM) + 1 120583L Maxima HMinus
Ez Mix + 8 120583L RNase free water
Invitrogen SuperScript ViloMaster Mix
M-MLV RTlowastRandom primer
10min-25∘C30min-42∘C5min-85∘C
4120583L SuperScript Vilo MasterMix (5x) + 11 120583LRNase free water
Quanta qScript cDNASynthesis Kit
M-MLV RTlowastMix OligodT
+ random primer
5min-22∘C30min-42∘C5min-85∘C
4 120583L qScript Reaction Mix (5x) + 1 120583L qScriptRTase + 10 120583L RNase free water
lowastMoloney Murine Leukemia ViruslowastlowastTotal volume of reaction is 20120583L containing 5120583L of RNA sample and 15 120583L of reaction mix All RT assays were performed on the Mastercycler Eppendorfthermal cyclerRTase reverse transcriptase
(b) Reactions mix and thermocycling conditions for the five qPCR SybrGreen kits (two-step)
Manufacturer qPCRSybrGreen kit Thermocycling conditions Reaction mixlowast
Bio-RadSsoFastEvaGreenSupermix
30 sec-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 20 120583L containing 2 120583L of cDNA and18 120583L of a reaction mix (1x Sso Fast master mix035 120583M [05 120583M] each of pan-Lyssavirus primers)
QiagenQuantiTectSYBR GreenPCR Kit
15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 25 120583L containing 2 120583L of cDNA and23 120583L of reaction mix (1x Quantitect Master mix05 120583M [06 120583M] each of pan-Lyssavirus primers)
FermentasMaxima SYBRGreen qPCRMaster Mix
10min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 25 120583L containing 2 120583L of cDNA and23 120583L of reaction mix (1x Maxima SYBR Green mastermix 04 120583M [1 120583M] each of pan-Lyssavirus primers)
Invitrogen
Express SYBRGreenER qPCR
SupermixUniversal
2min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 20 120583L containing 2 120583L of cDNA and18 120583L of reaction mix (1x Express SYBR GreenERSupermix 04 120583M [2 120583M] each of pan-Lyssavirus
primers) [50 nM of ROX dye]
Quanta Perfecta SYBRGreen SuperMix
3min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
Total volume of 25 120583L containing 2 120583L of cDNA and23120583L of reaction mix (1x Perfecta SYBR GreenSupermix 03 120583M [1 120583M] each of pan-Lyssavirus
primers)lowastThe PCR reaction protocol corresponds to PCR assays performed on the Rotor Gene Q MDx The concentrations of primers used on the real timethermocycler Mx3005P are shown in brackets [ ]
(c) Reactions mix and thermocycling conditions for the five qRT-PCR SYBR Green kits (one-step)
Manufacturer qRT-PCRSybrGreen kit Thermocycling conditions Reaction mixlowast
Qiagen QuantiTect SYBRGreen RT-PCR Kit
30min-50∘C 15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x QuantiTect SYBR Green master mix1 Quantitect RT mix 06 120583M [2120583M]
each of pan-Lyssavirus primers
4 BioMed Research International
(c) Continued
Manufacturer qRT-PCRSybrGreen kit Thermocycling conditions Reaction mixlowast
QiagenRotor Gene-1 stepRotor-Gene SYBRGreen RT-PCR Kit
30min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x Rotor-Gene master mix 1Rotor-Gene RT mix 1 120583M [2 120583M] each of
pan-Lyssavirus primers
Fermentas Verso 1-stepQRT-PCR Kit
30min-50∘C 15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x Verso SYBR Green master mix 5 RTenhancer 1 Verso enzyme mix 02 120583M[03 120583M] each of pan-Lyssavirus primers
Invitrogen
SuperScript IIIPlatinum SYBRGreen One-StepqRT-PCR Kit
10min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x SuperScript III Platinium master mix2 enzyme mix 03 120583M [05120583M] each of
pan-Lyssavirus primers
QuantaqScript One-StepSYBR GreenRT-qPCR Kit
30min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x qScript master mix 2 qScript enzymemix 04 120583M [02 120583M] each of
pan-Lyssavirus primerslowastTotal volume of reaction is 25 120583L containing 2120583L of RNA sample and 23120583L of reactionmixThe PCR reaction protocol corresponds to PCR assays performedon the Rotor Gene Q MDx The concentrations of primers used on the real time thermocycler Mx3005P are shown in brackets [ ]
five suppliers were tested with the two-step method Thefollowing RT kits were compared Iscript (IScript cDNA syn-thesis kit Bio-Rad France) QuantiTect (Quantitect Reversetranscription kit Qiagen France) Maxima (Maxima HMinus First Strand cDNA synthesis kit Fermentas France)SS Vilo (Superscript Vilo Master Mix Invitrogen France)and qScript (qScript cDNA Synthesis kit Quanta France)
(b) Two-Step SYBR Green PCR Kits (Table 1(b)) Differenttwo-step qPCR kits were tested SsoFast (SsoFast EvagreenSuper mix kit Bio-Rad France) QuantiTect (QuantiTectSYBR Green PCR kit Qiagen France) Maxima (MaximaSYBR Green qPCR master mix Fermentas France) Express(Express SYBR GreenER qPCR Supermix universal Invitro-gen France) and Perfecta (Perfecta SYBR Green SupermixQuanta France)
(2) Two-Step RT-qPCR Analysis Seven 1 10 serial dilutions(ie 106 to 1 copie120583L) of the in vitro EBLV-1 RNA transcriptwere prepared in RNAse free water then subjected to reversetranscription using the five different RT master mixes AllRT kits were optimised with regard to the time and the tem-perature of incubation (Table 1(a)) Complementary cDNAswere generated from 5120583L of each dilution of EBLV-1 RNArandomprimer oligo dT or amix of oligo dTrandomprimerincluded in each tested kit according to the recommendationsof themanufacturersThe cycling conditions and the reactionprotocol for each tested RT kit are detailed in Table 1(a)Ther-mocycling was performed on the Mastercycler Eppendorfthermal cycler (Eppendorf France)
Real-time PCRs were run on the thermocyclers RotorGene QMDx (Qiagen France) and Mx3005P (Agilent Tech-nologies France) The concentrations of the reverse (N165-146) and forward (JW12) primers were optimised for eachtested kit At least three different concentrations were exam-ined that is the one recommended by the manufacturerone below and one above the recommended primer concen-tration using the qPCR conditions described in Table 1(b)The optimised concentration of primers is recorded for each
tested qPCR kits in Table 1(b) Reactions were performed intriplicate in a total volume of 20 120583L or 25 120583L following themanufacturerrsquos instructions (Table 1(b))
The following thermal profile was used PCR initialactivation step at 95∘C for 30 sec (SsoFast) 15min (Quan-tiTect) 10min (Maxima) 2min (Express) or 3min (Per-fecta) according to themanufacturerrsquos recommendationsThecycler conditions performed as previously described byHay-man et al [19] were the same for all assays 45 cycles of three-step cycling consisting of denaturation at 94∘C for 30 secannealing at 55∘C for 30 sec and extension at 72∘C for 30 sec
233 One-Step qRT-PCR Assays
(1) Commercial qRT-PCR Kits (Table 1(c)) Five commer-cial one-step kits provided by four suppliers were testedQuantiTect-1 step (QuantiTect SYBR Green RT-PCR kitQiagen France) RotorGene-1 step (Rotor-Gene SYBRGreenRT-PCR kit Qiagen France) Verso-1 step (Verso SYBRGreen one-step qRT-PCR kit Fermentas France) SSIIIPlatinium-1 step (SuperScript III Platinium SYBR GreenOne-step qRT-PCR kit Invitrogen France) and qScript-1step (qScript One step SYBR Green QRT-PCR kit QuantaFrance)
(2) One-Step qRT-PCR Analysis To compare the five mastermixes the assays were performed in triplicate using seventenfold dilutions (ie 106 to 1 copies120583L) of EBLV-1 RNAPCR was performed using a total volume of 25 120583L containing2 120583L of serial dilutions of EBLV-1 RNA and the master mixcomponents (119881 = 23 120583L) from each representative kit whichwere prepared according to the respective manufacturerrsquosinstructions (Table 1(c)) The concentrations of the reverse(N165-146) and forward (JW12) primers were optimised foreach tested kit (Table 1(c)) At least three different con-centrations were examined the one recommended by themanufacturer one below and one above the recommendedprimer concentration (Table 1(b))
BioMed Research International 5
Real-time RT-PCR assays were performed on the ther-mocycler Rotor Gene Q MDx and Mx3005P using the samethermocycling conditions for the five kits (ie 45 cycles of30 sec-94∘C 30 sec-55∘C and 30 sec-72∘C) For each testedkit the DNA polymerase activation step was performed asrecommended by the supplier (Table 1(c))
234 Data Collection and Analysis PCR efficiency and coef-ficient of determination (1198772) values were calculated by thesoftware Rotor Gene Q Series and Mx3005Pro respectively
Negative and positive controls were included in eachassay in which a threshold setting (Ct) of 003 was used as thereference for the Rotor Gene QMDx thermocycler A thresh-old setting of 300 was used as the reference for the Mx3005Pthermocycler for all tested kits except for one two-step kit(Express) for which a threshold setting of 005 was used
24 Determination of the Performance of the Method Theperformance of the method was determined for all testedkits following OIE recommendations [20] and those of theFrench Agency for Standard Operating Procedure for PCRexperimental design [21] Performance testingwas conductedfor the two real-time platforms
241 Determination of PCR Efficiency A 106 to 103 copies120583Ldilution range corresponds to the linear dynamic range forthe ten tested kits To determine the PCR efficiency foreach tested kit a standard curve was plotted based on Ctvalues obtained from samples containing 106 to 103 copies120583Lagainst the log of the estimated RNA copy number in thesample The coefficient of determination (1198772) was calculatedand considered as suitable when not lower than 099 Theslope of the standard curves was used for the determinationof PCR efficiency (119864 = 10[minus1slope] minus 1) The efficiency wasconsidered satisfactory when it is not lower than 90 and notabove 110 [22]
242 Determination of the Limit of Detection at 95 Thelimit of detection at 95 (LOD 95) which corresponds tothe lowest RNA concentration that could be detected with95 confidence was calculated using the inverse standardnormal distribution calculation [21] The LOD 95 wascalculated using the Probit analysis based on the results ofthree runs of eight repetitions of six dilutions each of EBLV-1RNA in RNase-free water (119899 = 144 samples tested) The sixdilutions (cDNA and RNA) were selected surrounding thelimit of detection of real-time PCR assays
On the Rotor Gene Q MDx gene cycler 103 to 25 cop-iessample were tested with the kit Rotor Gene-1 step 103to 50 copiessample for qScript-1 stepSSIII Platinium-1 step500 to 10 copiessample for Verso-1 step 50 to 1 copiessamplefor Quantitect-1 step 250 to 5 copiessample for Perfecta 250to 5 copiessample for QuantiTect 102 to 25 copiessamplefor Maxima 100 to 05 copiessample for Sso Fast and 100 to05 copiessample for Express
On Mx3005P cycler 2500 to 100 copiessample weretested with the kit qScript-1 step 250 to 5 copiessample forSSIII Platinium-1 stepVerso-1 step 50 to 1 copiessample forRotor Gene-1 stepQuantitect-1 step 500 to 10 copiessample
for QuantiTect 250 to 5 copies120583L for MaximaSso FastPer-fecta 250 to 5 copiessample for Perfecta and 100 to 25 cop-iessample for Express
Three independent assays were undertaken under identi-cal laboratory conditions
243 Evaluation of Reproducibility To evaluate the repro-ducibility of the ten kits inter- and intraspecific assay repro-ducibility were assessed using the coefficient of variation(CV) of theCt values for the lowest RNAconcentration show-ing 100 positive results The intra-assay repeatability wasdetermined using three independent runs of eight replicatesof EBLV-1 (119899 = 24 samples tested) The interassay repeata-bility was assessed for the same serial replicates in threeindependent assays using identical laboratory conditionsCVs expressed as percentages were obtained by dividing thestandard deviation of each tested sample by the mean of thecycle threshold values and multiplying that result by 100
3 Results
31 Differences in RNADetection Levels Using Two-StepAssays
311 Reverse Transcription Kits Of the five reverse transcrip-tion kits tested in combination with the SSoFast kit for cDNAamplification the SuperScript Vilo yielded greater sensitivityand resulted in a detection limit of 10 copies120583L (3 positivesamples out of 3 tested) The efficiency of amplification wasof 98The four other RT kits (ie kits Quantitect MaximaSS Vilo and qScript) resulted in a detection of 100 copies120583LThe amplification efficiency varied between 98 and 104The 119910-intercept for all reverse transcription kits tested incombination with the qPCR SsoFast kit varied between 3513and 3746
312 Two-Step PCR Kits All optimised two-step qPCRassays tested in combination with the Iscript RT showed sim-ilar sensitivity and enabled detection of at least 100 copies120583Lof EBLV-1 on the Rotor Gene Q MDx gene cycler Theefficiency of amplificationwhichwas satisfactory for all testedkits varied between 90 (ie kit Perfecta) and 99 (ie kitsSSo Fast Quantitect SG Maxima SG and Express) with an119910-intercept varying between 3603 and 3857
313 Comparison of Two-Step RT-qPCR Kits Provided bythe Same Manufacturer Table 2 details the results of thecomparison of the five two-step kits
(i) Rotor Gene Q MDx Of the five PCR kits tested incombination with the reverse transcriptases provided by thesame manufacturer one RT-qPCR assay (SuperScript Vilo incombination with qPCR Express kit) yielded greater sensitiv-ity and showed a detection of at least 10 copies120583L (Table 2)All amplicons investigated with the two-step method usingqPCR kits and RT kits from the same provider showedsatisfactory efficiency of amplification (100 plusmn 10) The 119910-intercept for all tested kits varied between 3473 and 3955
(ii) Mx3005P Of the five tested RT-qPCR kits one RT-qPCRassay (Maxima RTqPCRMaxima) yielded greater sensitivity
6 BioMed Research International
Table2Com
paris
onof
RT-qPC
RSybrGreen
kits(tw
o-ste
p)provided
bythes
amem
anufacturerRe
sults
offived
ifferentq
PCRkitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1RN
A
Testingof
different
RT-qPC
Rkits(tw
o-ste
p)provided
bythes
amem
anufacturerlowastlowastlowastlowast
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
IScriptR
T+qP
CRSsoF
ast
QuantiTectR
T+qP
CRQuantitect
Maxim
aRT+qP
CRMaxim
aSS
ViloRT
+qP
CREx
press
qScriptR
T+qP
CRPerfe
cta
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
5010
51658plusmn013
077
1947plusmn007
034
1650plusmn015
092
1501plusmn
005
035
1609plusmn003
019
1896plusmn004
021
2279plusmn026
115
1747plusmn009
052
1749plusmn010
054
1799plusmn029
159
105
5010
41993plusmn009
045
2289plusmn010
042
1950plusmn006
033
1821plusmn
005
026
1979plusmn003
013
2217plusmn006
025
2594plusmn009
035
2116plusmn029
138
2095plusmn003
015
2127plusmn015
072
104
5010
32328plusmn005
020
2589plusmn006
025
2295plusmn028
123
2168plusmn017
076
2318plusmn016
067
2575plusmn005
018
2960plusmn042
143
2429plusmn029
039
2440plusmn012
049
2455plusmn008
033
103
5010
22661plusmn
021
080
2964plusmn032
107
2643plusmn026
098
2481plusmn
005
019
2694plusmn008
030
2916plusmn023
080
3210plusmn019
060
2778plusmn030
109
2803plusmn074
263
2784plusmn012
042
102
5010
13045plusmn013
042
3220plusmn041
126
3059plusmn045
146
2833plusmn064
225
3140plusmn053
168
3238plusmn061
189
3512plusmn010
028
3046plusmn092
303
3105plusmn038
123
3135plusmn070
222
101
5010
023lowastlowast
13lowastlowast
23lowastlowast
3152plusmn13
3420
13lowastlowast
13lowastlowast
13lowastlowast
3276plusmn070
212
13lowastlowast
23lowastlowast
105
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
Efficiency
99
99
100
101
90
96
107
97
93
102
1198772
0999
0997
0997
0999
0999
099
9099
1099
7099
4099
8
119910-in
tercept
3665
3955
3631
3473
3767
3938
4183
3801
3850
3801
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883
num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 7
and showed a detection of at least 10 copies120583L All RT-qPCRassays showed satisfactory efficiency varying between 93 and107 with detection of at least 100 copies120583LThe 119910-interceptvaried between 3801 and 4183 for all tested kits
32 Differences in RNA Detection between the Five Kits TestedUsing the One-Step Method Table 3 details the results of thecomparison of the five one-step kits
(i) Rotor Gene Q MDx Three optimised qRT-PCR assaysrevealed poor sensitivity and enabled detection of at least103 copies120583L Of the five kits tested two kits (QuantiTect-1 step and Verso-1 step) yielded greater sensitivity with thedetection of at least 10 copies120583L (3 positive samples out of3 tested) All kits showed a satisfactory efficiency varyingbetween 94 and 104The119910-intercept varied between 3278and 3767 for the five tested one-step kits
A high presence of Primer-Dimer was shown for lowRNA concentrations for three kits SSIII Platinium-1 stepVerso-1 step and qScript-1 step
(ii) Mx3005P Of the five one-step kits tested using theMx3005P cycler three optimised one-step kits (ie Quanti-tect-1 step Rotor Gene-1 step and SSIII Platinium-1 step)revealed good sensitivity with the detection of at least10 copies120583L The two other optimised one-step assays (ieVerso-1 step and qScript-1 step) revealed poor sensitivitywith a detection of 102 and 103 copies120583L respectively Theamplification efficiencywas satisfactory for all kits (94ndash100)except for the qScript one-step kit (efficiency of 87) The 119910-intercept varied between 3680 and 4074 for all tested kitsexcept for the qScript kit (Intercept of 4074)
33 Comparison of Assay Performance
331 Limit of Detection at 95 for One-Step and Two-StepMethods The results of LOD 95 for the ten kits tested usingtwo-step and one-step methods are recorded in Table 4
(i) Rotor Gene Q MDx The results showed an LOD 95 fortwo-step and one-step kits ranging from 41 to 171 copies120583Land from 20 to 497 copies120583L respectively (Table 4) Ofthe five commercial master mixes tested using the two-stepqPCR the RT SuperScript Vilo in combination with theExpress kit yielded greater sensitivity with an LOD 95 of41 copies120583L Maxima in combination with the RT Maximaand Perfecta in combination with the RT qScript rankedsecond and third with detection of 78 and 88 copies120583Lrespectively followed by the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 110 and 171 copies120583L respectively
In contrast the one-step qRT-PCR kit Quantitect-1 stepyielded the best levels of detection with an LOD 95 of20 copies120583L The four other tested one-step kits yieldedpoor detection with an LOD 95 varying between 127 and497 copies120583L
(ii) Mx3005P The LOD 95 for one-step and two-step kitsranged from 15 to 981 copies120583L and 45 to 160 copies120583L
respectivelyOf the five commercialmastermixes tested usingthe two-step qPCR the RT SuperScript Vilo in combinationwith the Express kit yielded greater sensitivity with an LOD95 of 45 copies120583L Perfecta in combination with qScriptRT and Maxima in combination with RT Maxima rankedsecond and third with a detection of 67 and 76 copies120583Lrespectively followed by the the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 88 and 160 copies120583L respectively
Of the five tested one-step kits two kits (ie Rotorgene-1 step and Quantitect-1 step) yielded the best levelsof detection with an LOD 95 of 15 and 22 copies120583Lrespectively SSIII Platinium-1 step ranked third with an LOD95 of 50 copies120583L followed by Verso-1 step (LOD 95 of101 copies120583L) and qScript-1 step (981 copies120583L)
332 Reproducibility of One-Step and Two-Step MethodsTable 5 shows the analysis results of reproducibility of one-step and two-step assays
(i) Rotor Gene Q MDx For all optimised one-step or two-step PCR assays the interassay CVs were lower than 10demonstrating good reproducibility for all tested kits [23]The interassay CVs for the two-step kits and the one-step kitsranged from 174 to 295 and 135 to 427 respectively whilethe intra-assay CVs ranged from 135 to 35 and 079 to 672for two-step and one-step kits respectively (Tables 5(a) and5(b)) In analysing the intra-assay CVs a somewhat similarvariability was shown for two-step and one-step reactions
The interassay CVs for the most efficient two-step kit(ie SuperScript Vilo in combination with qPCR Express)and the most efficient one-step kit (ie Quantitect-1 step)ranged from 195 to 336 respectively while the intra-assayCVs ranged from 185 to 211 and 079 to 216 for two-step andone-step kits respectively (Tables 5(a) and 5(b))
(ii) Mx3005P For all optimised one-step or two-step PCRassays the interassay CVs were lower than 10 demonstrat-ing good reproducibility for all tested kitsThe interassay CVsfor the two-step and one-step kits ranged from 214 to 33and 084 to 45 respectively while the intra-assay CVs rangedfrom 123 to 422 and 038 to 47 for two-step and one-stepkits respectively
34 Practicability of the Different Assays Costs and TimeTable 6 shows a practicability assessment of the differentreal-time assays including a comparison of cost pipettingsteps and thermocycling times Total reaction times for theten real-time assays ranged from 2h42 to 3h08 for two-stepkits and 2h18 to 2h57 for one-step kits with the one-stepSSIII Platinium kit requiring the shortest time and two-stepMaxima kit the longest regardless the machine used Thekits were ranked from 1 to 10 based on the cost per PCRreaction in increasing order from C108 (least expensive =1) to C1225 (most expensive = 10) Overall of the 10 mastermix kits tested the one-step kits were cheaper than the two-step kits (Table 6) For the one-step kits qScript was theleast costly followed by the Rotor Gene-1 step Verso-1 stepSSIII Platinium-1 step and the Quantitect-1 step which was
8 BioMed Research International
Table3Com
paris
onof
qRT-PC
RSY
BRGreen
(one-step)
kits
Results
offived
ifferentq
RT-PCR
kitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1in
vitro
RNAtranscrip
tsTestingof
different
qRT-PC
Rkits(one-step)
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Quantitect-1ste
pRo
torG
ene-1step
Verso-1step
SSIIIP
latinium-1ste
pqScript-1
step
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
2010
61666plusmn008
045
1715plusmn005
029
1699plusmn006
038
1452plusmn019
032
1347plusmn003
019
1779plusmn024
132
1588plusmn022
136
1834plusmn032
172
1622plusmn004
022
1848plusmn004
020
105
2010
52006plusmn010
052
2062plusmn004
017
2040plusmn011
052
1790plusmn021
118
1655plusmn003
019
2113plusmn045
212
1942plusmn020
104
2160plusmn015
071
1964plusmn041
211
2238plusmn015
068
104
2010
42358plusmn014
058
2391plusmn
008
033
2382plusmn020
083
2143plusmn012
057
1986plusmn004
018
2460plusmn020
083
2287plusmn016
069
2517plusmn017
069
2308plusmn027
116
2611plusmn010
038
103
2010
32670plusmn024
091
2745plusmn028
101
2693plusmn003
011
2490plusmn023
092
2313plusmn016
067
2795plusmn026
093
2634plusmn033
124
2863plusmn030
106
2614plusmn010
038
2954plusmn025
086
102
2010
23042plusmn008
026
23lowastlowast
3019plusmn060
198
03lowastlowast
03lowastlowast
3198plusmn017
053
2977plusmn039
132
3174plusmn079
250
2893plusmn008
028
3107plusmn037
118
101
2010
13324plusmn029
087
13lowastlowast
3327plusmn15
3460
03lowastlowast
03lowastlowast
3434plusmn10
3299
3284plusmn044
133
23lowastlowast
2991plusmn
010
033
03lowastlowast
12
03lowastlowast
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
03lowastlowast
03lowastlowast
Efficiency
98
96
100
94
104
97
94
95
100
87
1198772
0998
0999
0999
0998
0999
099
6099
8099
7099
6099
8
119910-in
tercept
3689
3767
3699
3529
3278
3815
3680
3893
3893
4074
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 9
Table4Detectio
nlim
itof
PCR(LOD95)for
two-ste
pandon
e-ste
pSY
BRGreen
RT-PCR
Two-ste
pRT
-qPC
RIScriptR
T+qP
CRSsoF
ast
BioR
adQuantiTectR
T+qP
CRQuantitect
QIAGEN
Maxim
aRT+qP
CRMaxim
aFE
RMEN
TAS
SSViloRT
+qP
CREx
press
INVITRO
GEN
qScriptR
T+qP
CRPerfe
cta
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
11055
171
8678
3941
2188
4488
44160
8076
3845
2267
34One-stepqR
T-PC
RQuantitect-1ste
pQIAGEN
RotorG
ene-1step
QIAGEN
Verso-1step
FERM
ENTA
SSSIIIP
latinium-1ste
pIN
VITRO
GEN
qScript-1
step
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
2040
497
994
127
254
321
641
406
812
2244
1530
101
202
50100
981
1962
lowastAllLO
D95results
werec
arrie
dou
tusin
gProb
itmod
ellowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
10 BioMed Research International
Table5Analysis
ofreprod
ucibilityof
one-ste
pandtwo-ste
pRT
-PCR
(a)
show
sfor
each
two-ste
pkittheintra-a
ndinterassay
reprod
ucibilityforsixdilutio
nsof
EBLV
-1teste
deighttim
esin
each
ofthreeind
ependent
runs
(119899=144samples)a
nd(b)sho
wsthe
inter-andintra-assayreprod
ucibilityfora
llteste
don
e-ste
pkits
(a)2-ste
pmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
IScriptR
T+qP
CRSsoF
ast
100
3046plusmn041
135
83008plusmn057
190
82982plusmn041
138
83012plusmn046
154
3012plusmn052
174
503111plusmn093
299
63184plusmn10
1319
63080plusmn046
151
83125plusmn080
256
3120plusmn088
283
253249plusmn059
183
63160plusmn067
212
83127plusmn068
219
63179plusmn065
204
3177plusmn080
251
103238plusmn091
281
63269plusmn119
363
63197plusmn076
239
53235plusmn095
294
3237plusmn097
299
53314
mdash1
mdashmdash
03198plusmn005
015
33256plusmn005
015
3227plusmn058
181
05
3217
mdash1
mdashmdash
03211plusmn076
236
33214plusmn076
236
3213plusmn062
193
250
3071plusmn
049
161
830
70plusmn039
128
830
61plusmn
033
108
830
67plusmn041
132
3067plusmn040
129
100
3230plusmn074
228
83203plusmn090
281
83219plusmn040
124
83217plusmn068
211
3217plusmn069
214
5034
02plusmn093
273
63324plusmn048
144
83316plusmn093
287
83347plusmn079
235
3342plusmn085
255
2534
63plusmn10
2295
83378plusmn075
223
634
24plusmn088
258
634
21plusmn
089
259
3425plusmn093
272
103595plusmn11
2312
53526plusmn047
134
534
68plusmn00
63
180
63530plusmn074
209
3526plusmn090
256
53384plusmn062
184
3mdash
mdash0
mdashmdash
034
83plusmn060
171
3384plusmn062
184
QuantiTectR
T+qP
CRQuantitect
250
3137plusmn069
220
83145plusmn052
165
83122plusmn046
147
83135plusmn056
177
3135plusmn055
175
100
3246plusmn080
246
73272plusmn092
281
73390plusmn090
265
73303plusmn087
264
3303plusmn10
5318
503359plusmn060
179
43434plusmn232
675
53366plusmn079
235
63386plusmn12
4363
3387plusmn14
0413
253298plusmn074
224
43358plusmn118
351
53412plusmn207
608
43356plusmn13
3394
3356plusmn13
8410
103354plusmn018
054
33386plusmn061
180
23473plusmn062
179
23404plusmn047
138
3397plusmn065
192
5mdash
mdash0
3552
mdash1
mdashmdash
03552
mdash3552
mdash50
034
09plusmn036
135
836
37plusmn118
326
63297plusmn038
116
834
30plusmn15
419
234
30plusmn15
4448
250
3642plusmn046
643
83366plusmn080
239
83361plusmn
083
246
834
56plusmn19
7376
3456plusmn19
7569
100
3439plusmn234
257
834
60plusmn098
284
834
50plusmn091
265
834
50plusmn089
269
3450plusmn089
258
5036
28plusmn088
475
63507plusmn091
259
734
92plusmn10
4297
73538plusmn13
2344
3538plusmn13
2374
253597plusmn17
2230
536
28plusmn088
242
436
44plusmn078
214
536
23plusmn079
229
3623plusmn079
217
10mdash
mdash0
mdashmdash
03765plusmn079
210
23765plusmn079
210
3765plusmn079
210
BioMed Research International 11
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
Maxim
aRT
+qP
CRMaxim
a
100
3183plusmn084
263
3138plusmn022
070
3009plusmn041
137
311plusmn049
157
311plusmn092
295
503335plusmn10
9326
83342plusmn088
264
53112plusmn064
205
83263plusmn087
265
3251plusmn
140
432
253367plusmn088
261
53277plusmn112
340
63247plusmn072
221
73297plusmn090
274
3290plusmn099
302
103716plusmn464
1249
33400plusmn14
1416
43435plusmn13
3388
53517plusmn246
684
3494plusmn263
754
53544plusmn024
068
23865
mdash1
3458plusmn12
4357
33622plusmn074
212
3555plusmn17
6496
25
mdashmdash
03314
mdash1
3347plusmn17
6526
23330plusmn17
6526
3336plusmn12
6377
250
2928plusmn058
199
829
05plusmn038
130
829
04plusmn039
135
829
12plusmn045
155
2912plusmn045
156
100
3053plusmn056
185
830
17plusmn048
159
830
31plusmn
043
143
830
34plusmn050
162
3034plusmn050
164
503154plusmn12
5397
83188plusmn13
7429
83172plusmn056
177
83171plusmn
108
334
3171plusmn
108
340
253300plusmn115
348
63238plusmn079
243
73238plusmn082
254
73256plusmn092
282
3256plusmn092
281
103308plusmn064
193
33302plusmn13
8417
43340plusmn14
6438
43317plusmn115
349
3317plusmn11
5347
503303plusmn015
045
23355
mdash1
3299
mdash1
3315plusmn028
045
3315plusmn028
085
qScriptR
T+qP
CRPerfe
cta
250
2704plusmn024
088
82675plusmn026
098
82677plusmn024
090
82685plusmn025
092
2685plusmn027
101
100
3054plusmn054
176
82984plusmn054
182
83046plusmn10
7350
83028plusmn072
236
3028plusmn079
261
503203plusmn290
905
73072plusmn089
292
83078plusmn082
267
83118plusmn15
4488
3114plusmn17
7568
253250plusmn098
302
73241plusmn
133
410
73363plusmn216
642
53285plusmn14
9452
3276plusmn14
9456
103222plusmn081
252
53287plusmn046
139
33270plusmn056
171
23260plusmn061
187
3251plusmn
069
211
53278plusmn015
045
2mdash
mdash0
3356plusmn15
5461
23318plusmn085
253
3318plusmn10
0303
250
2947plusmn022
074
829
49plusmn054
183
729
82plusmn038
129
829
60plusmn041
129
2960plusmn041
139
100
3099plusmn10
9352
83114plusmn12
6404
83102plusmn052
169
83105plusmn096
308
3105plusmn096
311
503187plusmn060
188
83137plusmn057
180
83313plusmn11
2337
83212plusmn10
8235
3212plusmn10
8335
253335plusmn299
896
73249plusmn068
211
83271plusmn
087
265
83283plusmn17
2457
3283plusmn17
2524
103300plusmn049
147
53345plusmn018
053
23298plusmn074
225
43307plusmn054
142
3307plusmn054
165
53304plusmn10
1306
23303plusmn074
223
23359
mdash1
3314plusmn067
265
3314plusmn067
203
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
4 BioMed Research International
(c) Continued
Manufacturer qRT-PCRSybrGreen kit Thermocycling conditions Reaction mixlowast
QiagenRotor Gene-1 stepRotor-Gene SYBRGreen RT-PCR Kit
30min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x Rotor-Gene master mix 1Rotor-Gene RT mix 1 120583M [2 120583M] each of
pan-Lyssavirus primers
Fermentas Verso 1-stepQRT-PCR Kit
30min-50∘C 15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x Verso SYBR Green master mix 5 RTenhancer 1 Verso enzyme mix 02 120583M[03 120583M] each of pan-Lyssavirus primers
Invitrogen
SuperScript IIIPlatinum SYBRGreen One-StepqRT-PCR Kit
10min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x SuperScript III Platinium master mix2 enzyme mix 03 120583M [05120583M] each of
pan-Lyssavirus primers
QuantaqScript One-StepSYBR GreenRT-qPCR Kit
30min-50∘C 5min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C
Melt Curve (60∘C to 95∘C)
1x qScript master mix 2 qScript enzymemix 04 120583M [02 120583M] each of
pan-Lyssavirus primerslowastTotal volume of reaction is 25 120583L containing 2120583L of RNA sample and 23120583L of reactionmixThe PCR reaction protocol corresponds to PCR assays performedon the Rotor Gene Q MDx The concentrations of primers used on the real time thermocycler Mx3005P are shown in brackets [ ]
five suppliers were tested with the two-step method Thefollowing RT kits were compared Iscript (IScript cDNA syn-thesis kit Bio-Rad France) QuantiTect (Quantitect Reversetranscription kit Qiagen France) Maxima (Maxima HMinus First Strand cDNA synthesis kit Fermentas France)SS Vilo (Superscript Vilo Master Mix Invitrogen France)and qScript (qScript cDNA Synthesis kit Quanta France)
(b) Two-Step SYBR Green PCR Kits (Table 1(b)) Differenttwo-step qPCR kits were tested SsoFast (SsoFast EvagreenSuper mix kit Bio-Rad France) QuantiTect (QuantiTectSYBR Green PCR kit Qiagen France) Maxima (MaximaSYBR Green qPCR master mix Fermentas France) Express(Express SYBR GreenER qPCR Supermix universal Invitro-gen France) and Perfecta (Perfecta SYBR Green SupermixQuanta France)
(2) Two-Step RT-qPCR Analysis Seven 1 10 serial dilutions(ie 106 to 1 copie120583L) of the in vitro EBLV-1 RNA transcriptwere prepared in RNAse free water then subjected to reversetranscription using the five different RT master mixes AllRT kits were optimised with regard to the time and the tem-perature of incubation (Table 1(a)) Complementary cDNAswere generated from 5120583L of each dilution of EBLV-1 RNArandomprimer oligo dT or amix of oligo dTrandomprimerincluded in each tested kit according to the recommendationsof themanufacturersThe cycling conditions and the reactionprotocol for each tested RT kit are detailed in Table 1(a)Ther-mocycling was performed on the Mastercycler Eppendorfthermal cycler (Eppendorf France)
Real-time PCRs were run on the thermocyclers RotorGene QMDx (Qiagen France) and Mx3005P (Agilent Tech-nologies France) The concentrations of the reverse (N165-146) and forward (JW12) primers were optimised for eachtested kit At least three different concentrations were exam-ined that is the one recommended by the manufacturerone below and one above the recommended primer concen-tration using the qPCR conditions described in Table 1(b)The optimised concentration of primers is recorded for each
tested qPCR kits in Table 1(b) Reactions were performed intriplicate in a total volume of 20 120583L or 25 120583L following themanufacturerrsquos instructions (Table 1(b))
The following thermal profile was used PCR initialactivation step at 95∘C for 30 sec (SsoFast) 15min (Quan-tiTect) 10min (Maxima) 2min (Express) or 3min (Per-fecta) according to themanufacturerrsquos recommendationsThecycler conditions performed as previously described byHay-man et al [19] were the same for all assays 45 cycles of three-step cycling consisting of denaturation at 94∘C for 30 secannealing at 55∘C for 30 sec and extension at 72∘C for 30 sec
233 One-Step qRT-PCR Assays
(1) Commercial qRT-PCR Kits (Table 1(c)) Five commer-cial one-step kits provided by four suppliers were testedQuantiTect-1 step (QuantiTect SYBR Green RT-PCR kitQiagen France) RotorGene-1 step (Rotor-Gene SYBRGreenRT-PCR kit Qiagen France) Verso-1 step (Verso SYBRGreen one-step qRT-PCR kit Fermentas France) SSIIIPlatinium-1 step (SuperScript III Platinium SYBR GreenOne-step qRT-PCR kit Invitrogen France) and qScript-1step (qScript One step SYBR Green QRT-PCR kit QuantaFrance)
(2) One-Step qRT-PCR Analysis To compare the five mastermixes the assays were performed in triplicate using seventenfold dilutions (ie 106 to 1 copies120583L) of EBLV-1 RNAPCR was performed using a total volume of 25 120583L containing2 120583L of serial dilutions of EBLV-1 RNA and the master mixcomponents (119881 = 23 120583L) from each representative kit whichwere prepared according to the respective manufacturerrsquosinstructions (Table 1(c)) The concentrations of the reverse(N165-146) and forward (JW12) primers were optimised foreach tested kit (Table 1(c)) At least three different con-centrations were examined the one recommended by themanufacturer one below and one above the recommendedprimer concentration (Table 1(b))
BioMed Research International 5
Real-time RT-PCR assays were performed on the ther-mocycler Rotor Gene Q MDx and Mx3005P using the samethermocycling conditions for the five kits (ie 45 cycles of30 sec-94∘C 30 sec-55∘C and 30 sec-72∘C) For each testedkit the DNA polymerase activation step was performed asrecommended by the supplier (Table 1(c))
234 Data Collection and Analysis PCR efficiency and coef-ficient of determination (1198772) values were calculated by thesoftware Rotor Gene Q Series and Mx3005Pro respectively
Negative and positive controls were included in eachassay in which a threshold setting (Ct) of 003 was used as thereference for the Rotor Gene QMDx thermocycler A thresh-old setting of 300 was used as the reference for the Mx3005Pthermocycler for all tested kits except for one two-step kit(Express) for which a threshold setting of 005 was used
24 Determination of the Performance of the Method Theperformance of the method was determined for all testedkits following OIE recommendations [20] and those of theFrench Agency for Standard Operating Procedure for PCRexperimental design [21] Performance testingwas conductedfor the two real-time platforms
241 Determination of PCR Efficiency A 106 to 103 copies120583Ldilution range corresponds to the linear dynamic range forthe ten tested kits To determine the PCR efficiency foreach tested kit a standard curve was plotted based on Ctvalues obtained from samples containing 106 to 103 copies120583Lagainst the log of the estimated RNA copy number in thesample The coefficient of determination (1198772) was calculatedand considered as suitable when not lower than 099 Theslope of the standard curves was used for the determinationof PCR efficiency (119864 = 10[minus1slope] minus 1) The efficiency wasconsidered satisfactory when it is not lower than 90 and notabove 110 [22]
242 Determination of the Limit of Detection at 95 Thelimit of detection at 95 (LOD 95) which corresponds tothe lowest RNA concentration that could be detected with95 confidence was calculated using the inverse standardnormal distribution calculation [21] The LOD 95 wascalculated using the Probit analysis based on the results ofthree runs of eight repetitions of six dilutions each of EBLV-1RNA in RNase-free water (119899 = 144 samples tested) The sixdilutions (cDNA and RNA) were selected surrounding thelimit of detection of real-time PCR assays
On the Rotor Gene Q MDx gene cycler 103 to 25 cop-iessample were tested with the kit Rotor Gene-1 step 103to 50 copiessample for qScript-1 stepSSIII Platinium-1 step500 to 10 copiessample for Verso-1 step 50 to 1 copiessamplefor Quantitect-1 step 250 to 5 copiessample for Perfecta 250to 5 copiessample for QuantiTect 102 to 25 copiessamplefor Maxima 100 to 05 copiessample for Sso Fast and 100 to05 copiessample for Express
On Mx3005P cycler 2500 to 100 copiessample weretested with the kit qScript-1 step 250 to 5 copiessample forSSIII Platinium-1 stepVerso-1 step 50 to 1 copiessample forRotor Gene-1 stepQuantitect-1 step 500 to 10 copiessample
for QuantiTect 250 to 5 copies120583L for MaximaSso FastPer-fecta 250 to 5 copiessample for Perfecta and 100 to 25 cop-iessample for Express
Three independent assays were undertaken under identi-cal laboratory conditions
243 Evaluation of Reproducibility To evaluate the repro-ducibility of the ten kits inter- and intraspecific assay repro-ducibility were assessed using the coefficient of variation(CV) of theCt values for the lowest RNAconcentration show-ing 100 positive results The intra-assay repeatability wasdetermined using three independent runs of eight replicatesof EBLV-1 (119899 = 24 samples tested) The interassay repeata-bility was assessed for the same serial replicates in threeindependent assays using identical laboratory conditionsCVs expressed as percentages were obtained by dividing thestandard deviation of each tested sample by the mean of thecycle threshold values and multiplying that result by 100
3 Results
31 Differences in RNADetection Levels Using Two-StepAssays
311 Reverse Transcription Kits Of the five reverse transcrip-tion kits tested in combination with the SSoFast kit for cDNAamplification the SuperScript Vilo yielded greater sensitivityand resulted in a detection limit of 10 copies120583L (3 positivesamples out of 3 tested) The efficiency of amplification wasof 98The four other RT kits (ie kits Quantitect MaximaSS Vilo and qScript) resulted in a detection of 100 copies120583LThe amplification efficiency varied between 98 and 104The 119910-intercept for all reverse transcription kits tested incombination with the qPCR SsoFast kit varied between 3513and 3746
312 Two-Step PCR Kits All optimised two-step qPCRassays tested in combination with the Iscript RT showed sim-ilar sensitivity and enabled detection of at least 100 copies120583Lof EBLV-1 on the Rotor Gene Q MDx gene cycler Theefficiency of amplificationwhichwas satisfactory for all testedkits varied between 90 (ie kit Perfecta) and 99 (ie kitsSSo Fast Quantitect SG Maxima SG and Express) with an119910-intercept varying between 3603 and 3857
313 Comparison of Two-Step RT-qPCR Kits Provided bythe Same Manufacturer Table 2 details the results of thecomparison of the five two-step kits
(i) Rotor Gene Q MDx Of the five PCR kits tested incombination with the reverse transcriptases provided by thesame manufacturer one RT-qPCR assay (SuperScript Vilo incombination with qPCR Express kit) yielded greater sensitiv-ity and showed a detection of at least 10 copies120583L (Table 2)All amplicons investigated with the two-step method usingqPCR kits and RT kits from the same provider showedsatisfactory efficiency of amplification (100 plusmn 10) The 119910-intercept for all tested kits varied between 3473 and 3955
(ii) Mx3005P Of the five tested RT-qPCR kits one RT-qPCRassay (Maxima RTqPCRMaxima) yielded greater sensitivity
6 BioMed Research International
Table2Com
paris
onof
RT-qPC
RSybrGreen
kits(tw
o-ste
p)provided
bythes
amem
anufacturerRe
sults
offived
ifferentq
PCRkitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1RN
A
Testingof
different
RT-qPC
Rkits(tw
o-ste
p)provided
bythes
amem
anufacturerlowastlowastlowastlowast
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
IScriptR
T+qP
CRSsoF
ast
QuantiTectR
T+qP
CRQuantitect
Maxim
aRT+qP
CRMaxim
aSS
ViloRT
+qP
CREx
press
qScriptR
T+qP
CRPerfe
cta
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
5010
51658plusmn013
077
1947plusmn007
034
1650plusmn015
092
1501plusmn
005
035
1609plusmn003
019
1896plusmn004
021
2279plusmn026
115
1747plusmn009
052
1749plusmn010
054
1799plusmn029
159
105
5010
41993plusmn009
045
2289plusmn010
042
1950plusmn006
033
1821plusmn
005
026
1979plusmn003
013
2217plusmn006
025
2594plusmn009
035
2116plusmn029
138
2095plusmn003
015
2127plusmn015
072
104
5010
32328plusmn005
020
2589plusmn006
025
2295plusmn028
123
2168plusmn017
076
2318plusmn016
067
2575plusmn005
018
2960plusmn042
143
2429plusmn029
039
2440plusmn012
049
2455plusmn008
033
103
5010
22661plusmn
021
080
2964plusmn032
107
2643plusmn026
098
2481plusmn
005
019
2694plusmn008
030
2916plusmn023
080
3210plusmn019
060
2778plusmn030
109
2803plusmn074
263
2784plusmn012
042
102
5010
13045plusmn013
042
3220plusmn041
126
3059plusmn045
146
2833plusmn064
225
3140plusmn053
168
3238plusmn061
189
3512plusmn010
028
3046plusmn092
303
3105plusmn038
123
3135plusmn070
222
101
5010
023lowastlowast
13lowastlowast
23lowastlowast
3152plusmn13
3420
13lowastlowast
13lowastlowast
13lowastlowast
3276plusmn070
212
13lowastlowast
23lowastlowast
105
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
Efficiency
99
99
100
101
90
96
107
97
93
102
1198772
0999
0997
0997
0999
0999
099
9099
1099
7099
4099
8
119910-in
tercept
3665
3955
3631
3473
3767
3938
4183
3801
3850
3801
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883
num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 7
and showed a detection of at least 10 copies120583L All RT-qPCRassays showed satisfactory efficiency varying between 93 and107 with detection of at least 100 copies120583LThe 119910-interceptvaried between 3801 and 4183 for all tested kits
32 Differences in RNA Detection between the Five Kits TestedUsing the One-Step Method Table 3 details the results of thecomparison of the five one-step kits
(i) Rotor Gene Q MDx Three optimised qRT-PCR assaysrevealed poor sensitivity and enabled detection of at least103 copies120583L Of the five kits tested two kits (QuantiTect-1 step and Verso-1 step) yielded greater sensitivity with thedetection of at least 10 copies120583L (3 positive samples out of3 tested) All kits showed a satisfactory efficiency varyingbetween 94 and 104The119910-intercept varied between 3278and 3767 for the five tested one-step kits
A high presence of Primer-Dimer was shown for lowRNA concentrations for three kits SSIII Platinium-1 stepVerso-1 step and qScript-1 step
(ii) Mx3005P Of the five one-step kits tested using theMx3005P cycler three optimised one-step kits (ie Quanti-tect-1 step Rotor Gene-1 step and SSIII Platinium-1 step)revealed good sensitivity with the detection of at least10 copies120583L The two other optimised one-step assays (ieVerso-1 step and qScript-1 step) revealed poor sensitivitywith a detection of 102 and 103 copies120583L respectively Theamplification efficiencywas satisfactory for all kits (94ndash100)except for the qScript one-step kit (efficiency of 87) The 119910-intercept varied between 3680 and 4074 for all tested kitsexcept for the qScript kit (Intercept of 4074)
33 Comparison of Assay Performance
331 Limit of Detection at 95 for One-Step and Two-StepMethods The results of LOD 95 for the ten kits tested usingtwo-step and one-step methods are recorded in Table 4
(i) Rotor Gene Q MDx The results showed an LOD 95 fortwo-step and one-step kits ranging from 41 to 171 copies120583Land from 20 to 497 copies120583L respectively (Table 4) Ofthe five commercial master mixes tested using the two-stepqPCR the RT SuperScript Vilo in combination with theExpress kit yielded greater sensitivity with an LOD 95 of41 copies120583L Maxima in combination with the RT Maximaand Perfecta in combination with the RT qScript rankedsecond and third with detection of 78 and 88 copies120583Lrespectively followed by the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 110 and 171 copies120583L respectively
In contrast the one-step qRT-PCR kit Quantitect-1 stepyielded the best levels of detection with an LOD 95 of20 copies120583L The four other tested one-step kits yieldedpoor detection with an LOD 95 varying between 127 and497 copies120583L
(ii) Mx3005P The LOD 95 for one-step and two-step kitsranged from 15 to 981 copies120583L and 45 to 160 copies120583L
respectivelyOf the five commercialmastermixes tested usingthe two-step qPCR the RT SuperScript Vilo in combinationwith the Express kit yielded greater sensitivity with an LOD95 of 45 copies120583L Perfecta in combination with qScriptRT and Maxima in combination with RT Maxima rankedsecond and third with a detection of 67 and 76 copies120583Lrespectively followed by the the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 88 and 160 copies120583L respectively
Of the five tested one-step kits two kits (ie Rotorgene-1 step and Quantitect-1 step) yielded the best levelsof detection with an LOD 95 of 15 and 22 copies120583Lrespectively SSIII Platinium-1 step ranked third with an LOD95 of 50 copies120583L followed by Verso-1 step (LOD 95 of101 copies120583L) and qScript-1 step (981 copies120583L)
332 Reproducibility of One-Step and Two-Step MethodsTable 5 shows the analysis results of reproducibility of one-step and two-step assays
(i) Rotor Gene Q MDx For all optimised one-step or two-step PCR assays the interassay CVs were lower than 10demonstrating good reproducibility for all tested kits [23]The interassay CVs for the two-step kits and the one-step kitsranged from 174 to 295 and 135 to 427 respectively whilethe intra-assay CVs ranged from 135 to 35 and 079 to 672for two-step and one-step kits respectively (Tables 5(a) and5(b)) In analysing the intra-assay CVs a somewhat similarvariability was shown for two-step and one-step reactions
The interassay CVs for the most efficient two-step kit(ie SuperScript Vilo in combination with qPCR Express)and the most efficient one-step kit (ie Quantitect-1 step)ranged from 195 to 336 respectively while the intra-assayCVs ranged from 185 to 211 and 079 to 216 for two-step andone-step kits respectively (Tables 5(a) and 5(b))
(ii) Mx3005P For all optimised one-step or two-step PCRassays the interassay CVs were lower than 10 demonstrat-ing good reproducibility for all tested kitsThe interassay CVsfor the two-step and one-step kits ranged from 214 to 33and 084 to 45 respectively while the intra-assay CVs rangedfrom 123 to 422 and 038 to 47 for two-step and one-stepkits respectively
34 Practicability of the Different Assays Costs and TimeTable 6 shows a practicability assessment of the differentreal-time assays including a comparison of cost pipettingsteps and thermocycling times Total reaction times for theten real-time assays ranged from 2h42 to 3h08 for two-stepkits and 2h18 to 2h57 for one-step kits with the one-stepSSIII Platinium kit requiring the shortest time and two-stepMaxima kit the longest regardless the machine used Thekits were ranked from 1 to 10 based on the cost per PCRreaction in increasing order from C108 (least expensive =1) to C1225 (most expensive = 10) Overall of the 10 mastermix kits tested the one-step kits were cheaper than the two-step kits (Table 6) For the one-step kits qScript was theleast costly followed by the Rotor Gene-1 step Verso-1 stepSSIII Platinium-1 step and the Quantitect-1 step which was
8 BioMed Research International
Table3Com
paris
onof
qRT-PC
RSY
BRGreen
(one-step)
kits
Results
offived
ifferentq
RT-PCR
kitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1in
vitro
RNAtranscrip
tsTestingof
different
qRT-PC
Rkits(one-step)
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Quantitect-1ste
pRo
torG
ene-1step
Verso-1step
SSIIIP
latinium-1ste
pqScript-1
step
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
2010
61666plusmn008
045
1715plusmn005
029
1699plusmn006
038
1452plusmn019
032
1347plusmn003
019
1779plusmn024
132
1588plusmn022
136
1834plusmn032
172
1622plusmn004
022
1848plusmn004
020
105
2010
52006plusmn010
052
2062plusmn004
017
2040plusmn011
052
1790plusmn021
118
1655plusmn003
019
2113plusmn045
212
1942plusmn020
104
2160plusmn015
071
1964plusmn041
211
2238plusmn015
068
104
2010
42358plusmn014
058
2391plusmn
008
033
2382plusmn020
083
2143plusmn012
057
1986plusmn004
018
2460plusmn020
083
2287plusmn016
069
2517plusmn017
069
2308plusmn027
116
2611plusmn010
038
103
2010
32670plusmn024
091
2745plusmn028
101
2693plusmn003
011
2490plusmn023
092
2313plusmn016
067
2795plusmn026
093
2634plusmn033
124
2863plusmn030
106
2614plusmn010
038
2954plusmn025
086
102
2010
23042plusmn008
026
23lowastlowast
3019plusmn060
198
03lowastlowast
03lowastlowast
3198plusmn017
053
2977plusmn039
132
3174plusmn079
250
2893plusmn008
028
3107plusmn037
118
101
2010
13324plusmn029
087
13lowastlowast
3327plusmn15
3460
03lowastlowast
03lowastlowast
3434plusmn10
3299
3284plusmn044
133
23lowastlowast
2991plusmn
010
033
03lowastlowast
12
03lowastlowast
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
03lowastlowast
03lowastlowast
Efficiency
98
96
100
94
104
97
94
95
100
87
1198772
0998
0999
0999
0998
0999
099
6099
8099
7099
6099
8
119910-in
tercept
3689
3767
3699
3529
3278
3815
3680
3893
3893
4074
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 9
Table4Detectio
nlim
itof
PCR(LOD95)for
two-ste
pandon
e-ste
pSY
BRGreen
RT-PCR
Two-ste
pRT
-qPC
RIScriptR
T+qP
CRSsoF
ast
BioR
adQuantiTectR
T+qP
CRQuantitect
QIAGEN
Maxim
aRT+qP
CRMaxim
aFE
RMEN
TAS
SSViloRT
+qP
CREx
press
INVITRO
GEN
qScriptR
T+qP
CRPerfe
cta
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
11055
171
8678
3941
2188
4488
44160
8076
3845
2267
34One-stepqR
T-PC
RQuantitect-1ste
pQIAGEN
RotorG
ene-1step
QIAGEN
Verso-1step
FERM
ENTA
SSSIIIP
latinium-1ste
pIN
VITRO
GEN
qScript-1
step
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
2040
497
994
127
254
321
641
406
812
2244
1530
101
202
50100
981
1962
lowastAllLO
D95results
werec
arrie
dou
tusin
gProb
itmod
ellowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
10 BioMed Research International
Table5Analysis
ofreprod
ucibilityof
one-ste
pandtwo-ste
pRT
-PCR
(a)
show
sfor
each
two-ste
pkittheintra-a
ndinterassay
reprod
ucibilityforsixdilutio
nsof
EBLV
-1teste
deighttim
esin
each
ofthreeind
ependent
runs
(119899=144samples)a
nd(b)sho
wsthe
inter-andintra-assayreprod
ucibilityfora
llteste
don
e-ste
pkits
(a)2-ste
pmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
IScriptR
T+qP
CRSsoF
ast
100
3046plusmn041
135
83008plusmn057
190
82982plusmn041
138
83012plusmn046
154
3012plusmn052
174
503111plusmn093
299
63184plusmn10
1319
63080plusmn046
151
83125plusmn080
256
3120plusmn088
283
253249plusmn059
183
63160plusmn067
212
83127plusmn068
219
63179plusmn065
204
3177plusmn080
251
103238plusmn091
281
63269plusmn119
363
63197plusmn076
239
53235plusmn095
294
3237plusmn097
299
53314
mdash1
mdashmdash
03198plusmn005
015
33256plusmn005
015
3227plusmn058
181
05
3217
mdash1
mdashmdash
03211plusmn076
236
33214plusmn076
236
3213plusmn062
193
250
3071plusmn
049
161
830
70plusmn039
128
830
61plusmn
033
108
830
67plusmn041
132
3067plusmn040
129
100
3230plusmn074
228
83203plusmn090
281
83219plusmn040
124
83217plusmn068
211
3217plusmn069
214
5034
02plusmn093
273
63324plusmn048
144
83316plusmn093
287
83347plusmn079
235
3342plusmn085
255
2534
63plusmn10
2295
83378plusmn075
223
634
24plusmn088
258
634
21plusmn
089
259
3425plusmn093
272
103595plusmn11
2312
53526plusmn047
134
534
68plusmn00
63
180
63530plusmn074
209
3526plusmn090
256
53384plusmn062
184
3mdash
mdash0
mdashmdash
034
83plusmn060
171
3384plusmn062
184
QuantiTectR
T+qP
CRQuantitect
250
3137plusmn069
220
83145plusmn052
165
83122plusmn046
147
83135plusmn056
177
3135plusmn055
175
100
3246plusmn080
246
73272plusmn092
281
73390plusmn090
265
73303plusmn087
264
3303plusmn10
5318
503359plusmn060
179
43434plusmn232
675
53366plusmn079
235
63386plusmn12
4363
3387plusmn14
0413
253298plusmn074
224
43358plusmn118
351
53412plusmn207
608
43356plusmn13
3394
3356plusmn13
8410
103354plusmn018
054
33386plusmn061
180
23473plusmn062
179
23404plusmn047
138
3397plusmn065
192
5mdash
mdash0
3552
mdash1
mdashmdash
03552
mdash3552
mdash50
034
09plusmn036
135
836
37plusmn118
326
63297plusmn038
116
834
30plusmn15
419
234
30plusmn15
4448
250
3642plusmn046
643
83366plusmn080
239
83361plusmn
083
246
834
56plusmn19
7376
3456plusmn19
7569
100
3439plusmn234
257
834
60plusmn098
284
834
50plusmn091
265
834
50plusmn089
269
3450plusmn089
258
5036
28plusmn088
475
63507plusmn091
259
734
92plusmn10
4297
73538plusmn13
2344
3538plusmn13
2374
253597plusmn17
2230
536
28plusmn088
242
436
44plusmn078
214
536
23plusmn079
229
3623plusmn079
217
10mdash
mdash0
mdashmdash
03765plusmn079
210
23765plusmn079
210
3765plusmn079
210
BioMed Research International 11
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
Maxim
aRT
+qP
CRMaxim
a
100
3183plusmn084
263
3138plusmn022
070
3009plusmn041
137
311plusmn049
157
311plusmn092
295
503335plusmn10
9326
83342plusmn088
264
53112plusmn064
205
83263plusmn087
265
3251plusmn
140
432
253367plusmn088
261
53277plusmn112
340
63247plusmn072
221
73297plusmn090
274
3290plusmn099
302
103716plusmn464
1249
33400plusmn14
1416
43435plusmn13
3388
53517plusmn246
684
3494plusmn263
754
53544plusmn024
068
23865
mdash1
3458plusmn12
4357
33622plusmn074
212
3555plusmn17
6496
25
mdashmdash
03314
mdash1
3347plusmn17
6526
23330plusmn17
6526
3336plusmn12
6377
250
2928plusmn058
199
829
05plusmn038
130
829
04plusmn039
135
829
12plusmn045
155
2912plusmn045
156
100
3053plusmn056
185
830
17plusmn048
159
830
31plusmn
043
143
830
34plusmn050
162
3034plusmn050
164
503154plusmn12
5397
83188plusmn13
7429
83172plusmn056
177
83171plusmn
108
334
3171plusmn
108
340
253300plusmn115
348
63238plusmn079
243
73238plusmn082
254
73256plusmn092
282
3256plusmn092
281
103308plusmn064
193
33302plusmn13
8417
43340plusmn14
6438
43317plusmn115
349
3317plusmn11
5347
503303plusmn015
045
23355
mdash1
3299
mdash1
3315plusmn028
045
3315plusmn028
085
qScriptR
T+qP
CRPerfe
cta
250
2704plusmn024
088
82675plusmn026
098
82677plusmn024
090
82685plusmn025
092
2685plusmn027
101
100
3054plusmn054
176
82984plusmn054
182
83046plusmn10
7350
83028plusmn072
236
3028plusmn079
261
503203plusmn290
905
73072plusmn089
292
83078plusmn082
267
83118plusmn15
4488
3114plusmn17
7568
253250plusmn098
302
73241plusmn
133
410
73363plusmn216
642
53285plusmn14
9452
3276plusmn14
9456
103222plusmn081
252
53287plusmn046
139
33270plusmn056
171
23260plusmn061
187
3251plusmn
069
211
53278plusmn015
045
2mdash
mdash0
3356plusmn15
5461
23318plusmn085
253
3318plusmn10
0303
250
2947plusmn022
074
829
49plusmn054
183
729
82plusmn038
129
829
60plusmn041
129
2960plusmn041
139
100
3099plusmn10
9352
83114plusmn12
6404
83102plusmn052
169
83105plusmn096
308
3105plusmn096
311
503187plusmn060
188
83137plusmn057
180
83313plusmn11
2337
83212plusmn10
8235
3212plusmn10
8335
253335plusmn299
896
73249plusmn068
211
83271plusmn
087
265
83283plusmn17
2457
3283plusmn17
2524
103300plusmn049
147
53345plusmn018
053
23298plusmn074
225
43307plusmn054
142
3307plusmn054
165
53304plusmn10
1306
23303plusmn074
223
23359
mdash1
3314plusmn067
265
3314plusmn067
203
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 5
Real-time RT-PCR assays were performed on the ther-mocycler Rotor Gene Q MDx and Mx3005P using the samethermocycling conditions for the five kits (ie 45 cycles of30 sec-94∘C 30 sec-55∘C and 30 sec-72∘C) For each testedkit the DNA polymerase activation step was performed asrecommended by the supplier (Table 1(c))
234 Data Collection and Analysis PCR efficiency and coef-ficient of determination (1198772) values were calculated by thesoftware Rotor Gene Q Series and Mx3005Pro respectively
Negative and positive controls were included in eachassay in which a threshold setting (Ct) of 003 was used as thereference for the Rotor Gene QMDx thermocycler A thresh-old setting of 300 was used as the reference for the Mx3005Pthermocycler for all tested kits except for one two-step kit(Express) for which a threshold setting of 005 was used
24 Determination of the Performance of the Method Theperformance of the method was determined for all testedkits following OIE recommendations [20] and those of theFrench Agency for Standard Operating Procedure for PCRexperimental design [21] Performance testingwas conductedfor the two real-time platforms
241 Determination of PCR Efficiency A 106 to 103 copies120583Ldilution range corresponds to the linear dynamic range forthe ten tested kits To determine the PCR efficiency foreach tested kit a standard curve was plotted based on Ctvalues obtained from samples containing 106 to 103 copies120583Lagainst the log of the estimated RNA copy number in thesample The coefficient of determination (1198772) was calculatedand considered as suitable when not lower than 099 Theslope of the standard curves was used for the determinationof PCR efficiency (119864 = 10[minus1slope] minus 1) The efficiency wasconsidered satisfactory when it is not lower than 90 and notabove 110 [22]
242 Determination of the Limit of Detection at 95 Thelimit of detection at 95 (LOD 95) which corresponds tothe lowest RNA concentration that could be detected with95 confidence was calculated using the inverse standardnormal distribution calculation [21] The LOD 95 wascalculated using the Probit analysis based on the results ofthree runs of eight repetitions of six dilutions each of EBLV-1RNA in RNase-free water (119899 = 144 samples tested) The sixdilutions (cDNA and RNA) were selected surrounding thelimit of detection of real-time PCR assays
On the Rotor Gene Q MDx gene cycler 103 to 25 cop-iessample were tested with the kit Rotor Gene-1 step 103to 50 copiessample for qScript-1 stepSSIII Platinium-1 step500 to 10 copiessample for Verso-1 step 50 to 1 copiessamplefor Quantitect-1 step 250 to 5 copiessample for Perfecta 250to 5 copiessample for QuantiTect 102 to 25 copiessamplefor Maxima 100 to 05 copiessample for Sso Fast and 100 to05 copiessample for Express
On Mx3005P cycler 2500 to 100 copiessample weretested with the kit qScript-1 step 250 to 5 copiessample forSSIII Platinium-1 stepVerso-1 step 50 to 1 copiessample forRotor Gene-1 stepQuantitect-1 step 500 to 10 copiessample
for QuantiTect 250 to 5 copies120583L for MaximaSso FastPer-fecta 250 to 5 copiessample for Perfecta and 100 to 25 cop-iessample for Express
Three independent assays were undertaken under identi-cal laboratory conditions
243 Evaluation of Reproducibility To evaluate the repro-ducibility of the ten kits inter- and intraspecific assay repro-ducibility were assessed using the coefficient of variation(CV) of theCt values for the lowest RNAconcentration show-ing 100 positive results The intra-assay repeatability wasdetermined using three independent runs of eight replicatesof EBLV-1 (119899 = 24 samples tested) The interassay repeata-bility was assessed for the same serial replicates in threeindependent assays using identical laboratory conditionsCVs expressed as percentages were obtained by dividing thestandard deviation of each tested sample by the mean of thecycle threshold values and multiplying that result by 100
3 Results
31 Differences in RNADetection Levels Using Two-StepAssays
311 Reverse Transcription Kits Of the five reverse transcrip-tion kits tested in combination with the SSoFast kit for cDNAamplification the SuperScript Vilo yielded greater sensitivityand resulted in a detection limit of 10 copies120583L (3 positivesamples out of 3 tested) The efficiency of amplification wasof 98The four other RT kits (ie kits Quantitect MaximaSS Vilo and qScript) resulted in a detection of 100 copies120583LThe amplification efficiency varied between 98 and 104The 119910-intercept for all reverse transcription kits tested incombination with the qPCR SsoFast kit varied between 3513and 3746
312 Two-Step PCR Kits All optimised two-step qPCRassays tested in combination with the Iscript RT showed sim-ilar sensitivity and enabled detection of at least 100 copies120583Lof EBLV-1 on the Rotor Gene Q MDx gene cycler Theefficiency of amplificationwhichwas satisfactory for all testedkits varied between 90 (ie kit Perfecta) and 99 (ie kitsSSo Fast Quantitect SG Maxima SG and Express) with an119910-intercept varying between 3603 and 3857
313 Comparison of Two-Step RT-qPCR Kits Provided bythe Same Manufacturer Table 2 details the results of thecomparison of the five two-step kits
(i) Rotor Gene Q MDx Of the five PCR kits tested incombination with the reverse transcriptases provided by thesame manufacturer one RT-qPCR assay (SuperScript Vilo incombination with qPCR Express kit) yielded greater sensitiv-ity and showed a detection of at least 10 copies120583L (Table 2)All amplicons investigated with the two-step method usingqPCR kits and RT kits from the same provider showedsatisfactory efficiency of amplification (100 plusmn 10) The 119910-intercept for all tested kits varied between 3473 and 3955
(ii) Mx3005P Of the five tested RT-qPCR kits one RT-qPCRassay (Maxima RTqPCRMaxima) yielded greater sensitivity
6 BioMed Research International
Table2Com
paris
onof
RT-qPC
RSybrGreen
kits(tw
o-ste
p)provided
bythes
amem
anufacturerRe
sults
offived
ifferentq
PCRkitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1RN
A
Testingof
different
RT-qPC
Rkits(tw
o-ste
p)provided
bythes
amem
anufacturerlowastlowastlowastlowast
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
IScriptR
T+qP
CRSsoF
ast
QuantiTectR
T+qP
CRQuantitect
Maxim
aRT+qP
CRMaxim
aSS
ViloRT
+qP
CREx
press
qScriptR
T+qP
CRPerfe
cta
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
5010
51658plusmn013
077
1947plusmn007
034
1650plusmn015
092
1501plusmn
005
035
1609plusmn003
019
1896plusmn004
021
2279plusmn026
115
1747plusmn009
052
1749plusmn010
054
1799plusmn029
159
105
5010
41993plusmn009
045
2289plusmn010
042
1950plusmn006
033
1821plusmn
005
026
1979plusmn003
013
2217plusmn006
025
2594plusmn009
035
2116plusmn029
138
2095plusmn003
015
2127plusmn015
072
104
5010
32328plusmn005
020
2589plusmn006
025
2295plusmn028
123
2168plusmn017
076
2318plusmn016
067
2575plusmn005
018
2960plusmn042
143
2429plusmn029
039
2440plusmn012
049
2455plusmn008
033
103
5010
22661plusmn
021
080
2964plusmn032
107
2643plusmn026
098
2481plusmn
005
019
2694plusmn008
030
2916plusmn023
080
3210plusmn019
060
2778plusmn030
109
2803plusmn074
263
2784plusmn012
042
102
5010
13045plusmn013
042
3220plusmn041
126
3059plusmn045
146
2833plusmn064
225
3140plusmn053
168
3238plusmn061
189
3512plusmn010
028
3046plusmn092
303
3105plusmn038
123
3135plusmn070
222
101
5010
023lowastlowast
13lowastlowast
23lowastlowast
3152plusmn13
3420
13lowastlowast
13lowastlowast
13lowastlowast
3276plusmn070
212
13lowastlowast
23lowastlowast
105
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
Efficiency
99
99
100
101
90
96
107
97
93
102
1198772
0999
0997
0997
0999
0999
099
9099
1099
7099
4099
8
119910-in
tercept
3665
3955
3631
3473
3767
3938
4183
3801
3850
3801
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883
num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 7
and showed a detection of at least 10 copies120583L All RT-qPCRassays showed satisfactory efficiency varying between 93 and107 with detection of at least 100 copies120583LThe 119910-interceptvaried between 3801 and 4183 for all tested kits
32 Differences in RNA Detection between the Five Kits TestedUsing the One-Step Method Table 3 details the results of thecomparison of the five one-step kits
(i) Rotor Gene Q MDx Three optimised qRT-PCR assaysrevealed poor sensitivity and enabled detection of at least103 copies120583L Of the five kits tested two kits (QuantiTect-1 step and Verso-1 step) yielded greater sensitivity with thedetection of at least 10 copies120583L (3 positive samples out of3 tested) All kits showed a satisfactory efficiency varyingbetween 94 and 104The119910-intercept varied between 3278and 3767 for the five tested one-step kits
A high presence of Primer-Dimer was shown for lowRNA concentrations for three kits SSIII Platinium-1 stepVerso-1 step and qScript-1 step
(ii) Mx3005P Of the five one-step kits tested using theMx3005P cycler three optimised one-step kits (ie Quanti-tect-1 step Rotor Gene-1 step and SSIII Platinium-1 step)revealed good sensitivity with the detection of at least10 copies120583L The two other optimised one-step assays (ieVerso-1 step and qScript-1 step) revealed poor sensitivitywith a detection of 102 and 103 copies120583L respectively Theamplification efficiencywas satisfactory for all kits (94ndash100)except for the qScript one-step kit (efficiency of 87) The 119910-intercept varied between 3680 and 4074 for all tested kitsexcept for the qScript kit (Intercept of 4074)
33 Comparison of Assay Performance
331 Limit of Detection at 95 for One-Step and Two-StepMethods The results of LOD 95 for the ten kits tested usingtwo-step and one-step methods are recorded in Table 4
(i) Rotor Gene Q MDx The results showed an LOD 95 fortwo-step and one-step kits ranging from 41 to 171 copies120583Land from 20 to 497 copies120583L respectively (Table 4) Ofthe five commercial master mixes tested using the two-stepqPCR the RT SuperScript Vilo in combination with theExpress kit yielded greater sensitivity with an LOD 95 of41 copies120583L Maxima in combination with the RT Maximaand Perfecta in combination with the RT qScript rankedsecond and third with detection of 78 and 88 copies120583Lrespectively followed by the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 110 and 171 copies120583L respectively
In contrast the one-step qRT-PCR kit Quantitect-1 stepyielded the best levels of detection with an LOD 95 of20 copies120583L The four other tested one-step kits yieldedpoor detection with an LOD 95 varying between 127 and497 copies120583L
(ii) Mx3005P The LOD 95 for one-step and two-step kitsranged from 15 to 981 copies120583L and 45 to 160 copies120583L
respectivelyOf the five commercialmastermixes tested usingthe two-step qPCR the RT SuperScript Vilo in combinationwith the Express kit yielded greater sensitivity with an LOD95 of 45 copies120583L Perfecta in combination with qScriptRT and Maxima in combination with RT Maxima rankedsecond and third with a detection of 67 and 76 copies120583Lrespectively followed by the the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 88 and 160 copies120583L respectively
Of the five tested one-step kits two kits (ie Rotorgene-1 step and Quantitect-1 step) yielded the best levelsof detection with an LOD 95 of 15 and 22 copies120583Lrespectively SSIII Platinium-1 step ranked third with an LOD95 of 50 copies120583L followed by Verso-1 step (LOD 95 of101 copies120583L) and qScript-1 step (981 copies120583L)
332 Reproducibility of One-Step and Two-Step MethodsTable 5 shows the analysis results of reproducibility of one-step and two-step assays
(i) Rotor Gene Q MDx For all optimised one-step or two-step PCR assays the interassay CVs were lower than 10demonstrating good reproducibility for all tested kits [23]The interassay CVs for the two-step kits and the one-step kitsranged from 174 to 295 and 135 to 427 respectively whilethe intra-assay CVs ranged from 135 to 35 and 079 to 672for two-step and one-step kits respectively (Tables 5(a) and5(b)) In analysing the intra-assay CVs a somewhat similarvariability was shown for two-step and one-step reactions
The interassay CVs for the most efficient two-step kit(ie SuperScript Vilo in combination with qPCR Express)and the most efficient one-step kit (ie Quantitect-1 step)ranged from 195 to 336 respectively while the intra-assayCVs ranged from 185 to 211 and 079 to 216 for two-step andone-step kits respectively (Tables 5(a) and 5(b))
(ii) Mx3005P For all optimised one-step or two-step PCRassays the interassay CVs were lower than 10 demonstrat-ing good reproducibility for all tested kitsThe interassay CVsfor the two-step and one-step kits ranged from 214 to 33and 084 to 45 respectively while the intra-assay CVs rangedfrom 123 to 422 and 038 to 47 for two-step and one-stepkits respectively
34 Practicability of the Different Assays Costs and TimeTable 6 shows a practicability assessment of the differentreal-time assays including a comparison of cost pipettingsteps and thermocycling times Total reaction times for theten real-time assays ranged from 2h42 to 3h08 for two-stepkits and 2h18 to 2h57 for one-step kits with the one-stepSSIII Platinium kit requiring the shortest time and two-stepMaxima kit the longest regardless the machine used Thekits were ranked from 1 to 10 based on the cost per PCRreaction in increasing order from C108 (least expensive =1) to C1225 (most expensive = 10) Overall of the 10 mastermix kits tested the one-step kits were cheaper than the two-step kits (Table 6) For the one-step kits qScript was theleast costly followed by the Rotor Gene-1 step Verso-1 stepSSIII Platinium-1 step and the Quantitect-1 step which was
8 BioMed Research International
Table3Com
paris
onof
qRT-PC
RSY
BRGreen
(one-step)
kits
Results
offived
ifferentq
RT-PCR
kitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1in
vitro
RNAtranscrip
tsTestingof
different
qRT-PC
Rkits(one-step)
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Quantitect-1ste
pRo
torG
ene-1step
Verso-1step
SSIIIP
latinium-1ste
pqScript-1
step
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
2010
61666plusmn008
045
1715plusmn005
029
1699plusmn006
038
1452plusmn019
032
1347plusmn003
019
1779plusmn024
132
1588plusmn022
136
1834plusmn032
172
1622plusmn004
022
1848plusmn004
020
105
2010
52006plusmn010
052
2062plusmn004
017
2040plusmn011
052
1790plusmn021
118
1655plusmn003
019
2113plusmn045
212
1942plusmn020
104
2160plusmn015
071
1964plusmn041
211
2238plusmn015
068
104
2010
42358plusmn014
058
2391plusmn
008
033
2382plusmn020
083
2143plusmn012
057
1986plusmn004
018
2460plusmn020
083
2287plusmn016
069
2517plusmn017
069
2308plusmn027
116
2611plusmn010
038
103
2010
32670plusmn024
091
2745plusmn028
101
2693plusmn003
011
2490plusmn023
092
2313plusmn016
067
2795plusmn026
093
2634plusmn033
124
2863plusmn030
106
2614plusmn010
038
2954plusmn025
086
102
2010
23042plusmn008
026
23lowastlowast
3019plusmn060
198
03lowastlowast
03lowastlowast
3198plusmn017
053
2977plusmn039
132
3174plusmn079
250
2893plusmn008
028
3107plusmn037
118
101
2010
13324plusmn029
087
13lowastlowast
3327plusmn15
3460
03lowastlowast
03lowastlowast
3434plusmn10
3299
3284plusmn044
133
23lowastlowast
2991plusmn
010
033
03lowastlowast
12
03lowastlowast
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
03lowastlowast
03lowastlowast
Efficiency
98
96
100
94
104
97
94
95
100
87
1198772
0998
0999
0999
0998
0999
099
6099
8099
7099
6099
8
119910-in
tercept
3689
3767
3699
3529
3278
3815
3680
3893
3893
4074
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 9
Table4Detectio
nlim
itof
PCR(LOD95)for
two-ste
pandon
e-ste
pSY
BRGreen
RT-PCR
Two-ste
pRT
-qPC
RIScriptR
T+qP
CRSsoF
ast
BioR
adQuantiTectR
T+qP
CRQuantitect
QIAGEN
Maxim
aRT+qP
CRMaxim
aFE
RMEN
TAS
SSViloRT
+qP
CREx
press
INVITRO
GEN
qScriptR
T+qP
CRPerfe
cta
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
11055
171
8678
3941
2188
4488
44160
8076
3845
2267
34One-stepqR
T-PC
RQuantitect-1ste
pQIAGEN
RotorG
ene-1step
QIAGEN
Verso-1step
FERM
ENTA
SSSIIIP
latinium-1ste
pIN
VITRO
GEN
qScript-1
step
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
2040
497
994
127
254
321
641
406
812
2244
1530
101
202
50100
981
1962
lowastAllLO
D95results
werec
arrie
dou
tusin
gProb
itmod
ellowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
10 BioMed Research International
Table5Analysis
ofreprod
ucibilityof
one-ste
pandtwo-ste
pRT
-PCR
(a)
show
sfor
each
two-ste
pkittheintra-a
ndinterassay
reprod
ucibilityforsixdilutio
nsof
EBLV
-1teste
deighttim
esin
each
ofthreeind
ependent
runs
(119899=144samples)a
nd(b)sho
wsthe
inter-andintra-assayreprod
ucibilityfora
llteste
don
e-ste
pkits
(a)2-ste
pmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
IScriptR
T+qP
CRSsoF
ast
100
3046plusmn041
135
83008plusmn057
190
82982plusmn041
138
83012plusmn046
154
3012plusmn052
174
503111plusmn093
299
63184plusmn10
1319
63080plusmn046
151
83125plusmn080
256
3120plusmn088
283
253249plusmn059
183
63160plusmn067
212
83127plusmn068
219
63179plusmn065
204
3177plusmn080
251
103238plusmn091
281
63269plusmn119
363
63197plusmn076
239
53235plusmn095
294
3237plusmn097
299
53314
mdash1
mdashmdash
03198plusmn005
015
33256plusmn005
015
3227plusmn058
181
05
3217
mdash1
mdashmdash
03211plusmn076
236
33214plusmn076
236
3213plusmn062
193
250
3071plusmn
049
161
830
70plusmn039
128
830
61plusmn
033
108
830
67plusmn041
132
3067plusmn040
129
100
3230plusmn074
228
83203plusmn090
281
83219plusmn040
124
83217plusmn068
211
3217plusmn069
214
5034
02plusmn093
273
63324plusmn048
144
83316plusmn093
287
83347plusmn079
235
3342plusmn085
255
2534
63plusmn10
2295
83378plusmn075
223
634
24plusmn088
258
634
21plusmn
089
259
3425plusmn093
272
103595plusmn11
2312
53526plusmn047
134
534
68plusmn00
63
180
63530plusmn074
209
3526plusmn090
256
53384plusmn062
184
3mdash
mdash0
mdashmdash
034
83plusmn060
171
3384plusmn062
184
QuantiTectR
T+qP
CRQuantitect
250
3137plusmn069
220
83145plusmn052
165
83122plusmn046
147
83135plusmn056
177
3135plusmn055
175
100
3246plusmn080
246
73272plusmn092
281
73390plusmn090
265
73303plusmn087
264
3303plusmn10
5318
503359plusmn060
179
43434plusmn232
675
53366plusmn079
235
63386plusmn12
4363
3387plusmn14
0413
253298plusmn074
224
43358plusmn118
351
53412plusmn207
608
43356plusmn13
3394
3356plusmn13
8410
103354plusmn018
054
33386plusmn061
180
23473plusmn062
179
23404plusmn047
138
3397plusmn065
192
5mdash
mdash0
3552
mdash1
mdashmdash
03552
mdash3552
mdash50
034
09plusmn036
135
836
37plusmn118
326
63297plusmn038
116
834
30plusmn15
419
234
30plusmn15
4448
250
3642plusmn046
643
83366plusmn080
239
83361plusmn
083
246
834
56plusmn19
7376
3456plusmn19
7569
100
3439plusmn234
257
834
60plusmn098
284
834
50plusmn091
265
834
50plusmn089
269
3450plusmn089
258
5036
28plusmn088
475
63507plusmn091
259
734
92plusmn10
4297
73538plusmn13
2344
3538plusmn13
2374
253597plusmn17
2230
536
28plusmn088
242
436
44plusmn078
214
536
23plusmn079
229
3623plusmn079
217
10mdash
mdash0
mdashmdash
03765plusmn079
210
23765plusmn079
210
3765plusmn079
210
BioMed Research International 11
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
Maxim
aRT
+qP
CRMaxim
a
100
3183plusmn084
263
3138plusmn022
070
3009plusmn041
137
311plusmn049
157
311plusmn092
295
503335plusmn10
9326
83342plusmn088
264
53112plusmn064
205
83263plusmn087
265
3251plusmn
140
432
253367plusmn088
261
53277plusmn112
340
63247plusmn072
221
73297plusmn090
274
3290plusmn099
302
103716plusmn464
1249
33400plusmn14
1416
43435plusmn13
3388
53517plusmn246
684
3494plusmn263
754
53544plusmn024
068
23865
mdash1
3458plusmn12
4357
33622plusmn074
212
3555plusmn17
6496
25
mdashmdash
03314
mdash1
3347plusmn17
6526
23330plusmn17
6526
3336plusmn12
6377
250
2928plusmn058
199
829
05plusmn038
130
829
04plusmn039
135
829
12plusmn045
155
2912plusmn045
156
100
3053plusmn056
185
830
17plusmn048
159
830
31plusmn
043
143
830
34plusmn050
162
3034plusmn050
164
503154plusmn12
5397
83188plusmn13
7429
83172plusmn056
177
83171plusmn
108
334
3171plusmn
108
340
253300plusmn115
348
63238plusmn079
243
73238plusmn082
254
73256plusmn092
282
3256plusmn092
281
103308plusmn064
193
33302plusmn13
8417
43340plusmn14
6438
43317plusmn115
349
3317plusmn11
5347
503303plusmn015
045
23355
mdash1
3299
mdash1
3315plusmn028
045
3315plusmn028
085
qScriptR
T+qP
CRPerfe
cta
250
2704plusmn024
088
82675plusmn026
098
82677plusmn024
090
82685plusmn025
092
2685plusmn027
101
100
3054plusmn054
176
82984plusmn054
182
83046plusmn10
7350
83028plusmn072
236
3028plusmn079
261
503203plusmn290
905
73072plusmn089
292
83078plusmn082
267
83118plusmn15
4488
3114plusmn17
7568
253250plusmn098
302
73241plusmn
133
410
73363plusmn216
642
53285plusmn14
9452
3276plusmn14
9456
103222plusmn081
252
53287plusmn046
139
33270plusmn056
171
23260plusmn061
187
3251plusmn
069
211
53278plusmn015
045
2mdash
mdash0
3356plusmn15
5461
23318plusmn085
253
3318plusmn10
0303
250
2947plusmn022
074
829
49plusmn054
183
729
82plusmn038
129
829
60plusmn041
129
2960plusmn041
139
100
3099plusmn10
9352
83114plusmn12
6404
83102plusmn052
169
83105plusmn096
308
3105plusmn096
311
503187plusmn060
188
83137plusmn057
180
83313plusmn11
2337
83212plusmn10
8235
3212plusmn10
8335
253335plusmn299
896
73249plusmn068
211
83271plusmn
087
265
83283plusmn17
2457
3283plusmn17
2524
103300plusmn049
147
53345plusmn018
053
23298plusmn074
225
43307plusmn054
142
3307plusmn054
165
53304plusmn10
1306
23303plusmn074
223
23359
mdash1
3314plusmn067
265
3314plusmn067
203
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 BioMed Research International
Table2Com
paris
onof
RT-qPC
RSybrGreen
kits(tw
o-ste
p)provided
bythes
amem
anufacturerRe
sults
offived
ifferentq
PCRkitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1RN
A
Testingof
different
RT-qPC
Rkits(tw
o-ste
p)provided
bythes
amem
anufacturerlowastlowastlowastlowast
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
IScriptR
T+qP
CRSsoF
ast
QuantiTectR
T+qP
CRQuantitect
Maxim
aRT+qP
CRMaxim
aSS
ViloRT
+qP
CREx
press
qScriptR
T+qP
CRPerfe
cta
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
5010
51658plusmn013
077
1947plusmn007
034
1650plusmn015
092
1501plusmn
005
035
1609plusmn003
019
1896plusmn004
021
2279plusmn026
115
1747plusmn009
052
1749plusmn010
054
1799plusmn029
159
105
5010
41993plusmn009
045
2289plusmn010
042
1950plusmn006
033
1821plusmn
005
026
1979plusmn003
013
2217plusmn006
025
2594plusmn009
035
2116plusmn029
138
2095plusmn003
015
2127plusmn015
072
104
5010
32328plusmn005
020
2589plusmn006
025
2295plusmn028
123
2168plusmn017
076
2318plusmn016
067
2575plusmn005
018
2960plusmn042
143
2429plusmn029
039
2440plusmn012
049
2455plusmn008
033
103
5010
22661plusmn
021
080
2964plusmn032
107
2643plusmn026
098
2481plusmn
005
019
2694plusmn008
030
2916plusmn023
080
3210plusmn019
060
2778plusmn030
109
2803plusmn074
263
2784plusmn012
042
102
5010
13045plusmn013
042
3220plusmn041
126
3059plusmn045
146
2833plusmn064
225
3140plusmn053
168
3238plusmn061
189
3512plusmn010
028
3046plusmn092
303
3105plusmn038
123
3135plusmn070
222
101
5010
023lowastlowast
13lowastlowast
23lowastlowast
3152plusmn13
3420
13lowastlowast
13lowastlowast
13lowastlowast
3276plusmn070
212
13lowastlowast
23lowastlowast
105
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
Efficiency
99
99
100
101
90
96
107
97
93
102
1198772
0999
0997
0997
0999
0999
099
9099
1099
7099
4099
8
119910-in
tercept
3665
3955
3631
3473
3767
3938
4183
3801
3850
3801
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883
num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 7
and showed a detection of at least 10 copies120583L All RT-qPCRassays showed satisfactory efficiency varying between 93 and107 with detection of at least 100 copies120583LThe 119910-interceptvaried between 3801 and 4183 for all tested kits
32 Differences in RNA Detection between the Five Kits TestedUsing the One-Step Method Table 3 details the results of thecomparison of the five one-step kits
(i) Rotor Gene Q MDx Three optimised qRT-PCR assaysrevealed poor sensitivity and enabled detection of at least103 copies120583L Of the five kits tested two kits (QuantiTect-1 step and Verso-1 step) yielded greater sensitivity with thedetection of at least 10 copies120583L (3 positive samples out of3 tested) All kits showed a satisfactory efficiency varyingbetween 94 and 104The119910-intercept varied between 3278and 3767 for the five tested one-step kits
A high presence of Primer-Dimer was shown for lowRNA concentrations for three kits SSIII Platinium-1 stepVerso-1 step and qScript-1 step
(ii) Mx3005P Of the five one-step kits tested using theMx3005P cycler three optimised one-step kits (ie Quanti-tect-1 step Rotor Gene-1 step and SSIII Platinium-1 step)revealed good sensitivity with the detection of at least10 copies120583L The two other optimised one-step assays (ieVerso-1 step and qScript-1 step) revealed poor sensitivitywith a detection of 102 and 103 copies120583L respectively Theamplification efficiencywas satisfactory for all kits (94ndash100)except for the qScript one-step kit (efficiency of 87) The 119910-intercept varied between 3680 and 4074 for all tested kitsexcept for the qScript kit (Intercept of 4074)
33 Comparison of Assay Performance
331 Limit of Detection at 95 for One-Step and Two-StepMethods The results of LOD 95 for the ten kits tested usingtwo-step and one-step methods are recorded in Table 4
(i) Rotor Gene Q MDx The results showed an LOD 95 fortwo-step and one-step kits ranging from 41 to 171 copies120583Land from 20 to 497 copies120583L respectively (Table 4) Ofthe five commercial master mixes tested using the two-stepqPCR the RT SuperScript Vilo in combination with theExpress kit yielded greater sensitivity with an LOD 95 of41 copies120583L Maxima in combination with the RT Maximaand Perfecta in combination with the RT qScript rankedsecond and third with detection of 78 and 88 copies120583Lrespectively followed by the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 110 and 171 copies120583L respectively
In contrast the one-step qRT-PCR kit Quantitect-1 stepyielded the best levels of detection with an LOD 95 of20 copies120583L The four other tested one-step kits yieldedpoor detection with an LOD 95 varying between 127 and497 copies120583L
(ii) Mx3005P The LOD 95 for one-step and two-step kitsranged from 15 to 981 copies120583L and 45 to 160 copies120583L
respectivelyOf the five commercialmastermixes tested usingthe two-step qPCR the RT SuperScript Vilo in combinationwith the Express kit yielded greater sensitivity with an LOD95 of 45 copies120583L Perfecta in combination with qScriptRT and Maxima in combination with RT Maxima rankedsecond and third with a detection of 67 and 76 copies120583Lrespectively followed by the the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 88 and 160 copies120583L respectively
Of the five tested one-step kits two kits (ie Rotorgene-1 step and Quantitect-1 step) yielded the best levelsof detection with an LOD 95 of 15 and 22 copies120583Lrespectively SSIII Platinium-1 step ranked third with an LOD95 of 50 copies120583L followed by Verso-1 step (LOD 95 of101 copies120583L) and qScript-1 step (981 copies120583L)
332 Reproducibility of One-Step and Two-Step MethodsTable 5 shows the analysis results of reproducibility of one-step and two-step assays
(i) Rotor Gene Q MDx For all optimised one-step or two-step PCR assays the interassay CVs were lower than 10demonstrating good reproducibility for all tested kits [23]The interassay CVs for the two-step kits and the one-step kitsranged from 174 to 295 and 135 to 427 respectively whilethe intra-assay CVs ranged from 135 to 35 and 079 to 672for two-step and one-step kits respectively (Tables 5(a) and5(b)) In analysing the intra-assay CVs a somewhat similarvariability was shown for two-step and one-step reactions
The interassay CVs for the most efficient two-step kit(ie SuperScript Vilo in combination with qPCR Express)and the most efficient one-step kit (ie Quantitect-1 step)ranged from 195 to 336 respectively while the intra-assayCVs ranged from 185 to 211 and 079 to 216 for two-step andone-step kits respectively (Tables 5(a) and 5(b))
(ii) Mx3005P For all optimised one-step or two-step PCRassays the interassay CVs were lower than 10 demonstrat-ing good reproducibility for all tested kitsThe interassay CVsfor the two-step and one-step kits ranged from 214 to 33and 084 to 45 respectively while the intra-assay CVs rangedfrom 123 to 422 and 038 to 47 for two-step and one-stepkits respectively
34 Practicability of the Different Assays Costs and TimeTable 6 shows a practicability assessment of the differentreal-time assays including a comparison of cost pipettingsteps and thermocycling times Total reaction times for theten real-time assays ranged from 2h42 to 3h08 for two-stepkits and 2h18 to 2h57 for one-step kits with the one-stepSSIII Platinium kit requiring the shortest time and two-stepMaxima kit the longest regardless the machine used Thekits were ranked from 1 to 10 based on the cost per PCRreaction in increasing order from C108 (least expensive =1) to C1225 (most expensive = 10) Overall of the 10 mastermix kits tested the one-step kits were cheaper than the two-step kits (Table 6) For the one-step kits qScript was theleast costly followed by the Rotor Gene-1 step Verso-1 stepSSIII Platinium-1 step and the Quantitect-1 step which was
8 BioMed Research International
Table3Com
paris
onof
qRT-PC
RSY
BRGreen
(one-step)
kits
Results
offived
ifferentq
RT-PCR
kitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1in
vitro
RNAtranscrip
tsTestingof
different
qRT-PC
Rkits(one-step)
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Quantitect-1ste
pRo
torG
ene-1step
Verso-1step
SSIIIP
latinium-1ste
pqScript-1
step
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
2010
61666plusmn008
045
1715plusmn005
029
1699plusmn006
038
1452plusmn019
032
1347plusmn003
019
1779plusmn024
132
1588plusmn022
136
1834plusmn032
172
1622plusmn004
022
1848plusmn004
020
105
2010
52006plusmn010
052
2062plusmn004
017
2040plusmn011
052
1790plusmn021
118
1655plusmn003
019
2113plusmn045
212
1942plusmn020
104
2160plusmn015
071
1964plusmn041
211
2238plusmn015
068
104
2010
42358plusmn014
058
2391plusmn
008
033
2382plusmn020
083
2143plusmn012
057
1986plusmn004
018
2460plusmn020
083
2287plusmn016
069
2517plusmn017
069
2308plusmn027
116
2611plusmn010
038
103
2010
32670plusmn024
091
2745plusmn028
101
2693plusmn003
011
2490plusmn023
092
2313plusmn016
067
2795plusmn026
093
2634plusmn033
124
2863plusmn030
106
2614plusmn010
038
2954plusmn025
086
102
2010
23042plusmn008
026
23lowastlowast
3019plusmn060
198
03lowastlowast
03lowastlowast
3198plusmn017
053
2977plusmn039
132
3174plusmn079
250
2893plusmn008
028
3107plusmn037
118
101
2010
13324plusmn029
087
13lowastlowast
3327plusmn15
3460
03lowastlowast
03lowastlowast
3434plusmn10
3299
3284plusmn044
133
23lowastlowast
2991plusmn
010
033
03lowastlowast
12
03lowastlowast
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
03lowastlowast
03lowastlowast
Efficiency
98
96
100
94
104
97
94
95
100
87
1198772
0998
0999
0999
0998
0999
099
6099
8099
7099
6099
8
119910-in
tercept
3689
3767
3699
3529
3278
3815
3680
3893
3893
4074
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 9
Table4Detectio
nlim
itof
PCR(LOD95)for
two-ste
pandon
e-ste
pSY
BRGreen
RT-PCR
Two-ste
pRT
-qPC
RIScriptR
T+qP
CRSsoF
ast
BioR
adQuantiTectR
T+qP
CRQuantitect
QIAGEN
Maxim
aRT+qP
CRMaxim
aFE
RMEN
TAS
SSViloRT
+qP
CREx
press
INVITRO
GEN
qScriptR
T+qP
CRPerfe
cta
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
11055
171
8678
3941
2188
4488
44160
8076
3845
2267
34One-stepqR
T-PC
RQuantitect-1ste
pQIAGEN
RotorG
ene-1step
QIAGEN
Verso-1step
FERM
ENTA
SSSIIIP
latinium-1ste
pIN
VITRO
GEN
qScript-1
step
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
2040
497
994
127
254
321
641
406
812
2244
1530
101
202
50100
981
1962
lowastAllLO
D95results
werec
arrie
dou
tusin
gProb
itmod
ellowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
10 BioMed Research International
Table5Analysis
ofreprod
ucibilityof
one-ste
pandtwo-ste
pRT
-PCR
(a)
show
sfor
each
two-ste
pkittheintra-a
ndinterassay
reprod
ucibilityforsixdilutio
nsof
EBLV
-1teste
deighttim
esin
each
ofthreeind
ependent
runs
(119899=144samples)a
nd(b)sho
wsthe
inter-andintra-assayreprod
ucibilityfora
llteste
don
e-ste
pkits
(a)2-ste
pmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
IScriptR
T+qP
CRSsoF
ast
100
3046plusmn041
135
83008plusmn057
190
82982plusmn041
138
83012plusmn046
154
3012plusmn052
174
503111plusmn093
299
63184plusmn10
1319
63080plusmn046
151
83125plusmn080
256
3120plusmn088
283
253249plusmn059
183
63160plusmn067
212
83127plusmn068
219
63179plusmn065
204
3177plusmn080
251
103238plusmn091
281
63269plusmn119
363
63197plusmn076
239
53235plusmn095
294
3237plusmn097
299
53314
mdash1
mdashmdash
03198plusmn005
015
33256plusmn005
015
3227plusmn058
181
05
3217
mdash1
mdashmdash
03211plusmn076
236
33214plusmn076
236
3213plusmn062
193
250
3071plusmn
049
161
830
70plusmn039
128
830
61plusmn
033
108
830
67plusmn041
132
3067plusmn040
129
100
3230plusmn074
228
83203plusmn090
281
83219plusmn040
124
83217plusmn068
211
3217plusmn069
214
5034
02plusmn093
273
63324plusmn048
144
83316plusmn093
287
83347plusmn079
235
3342plusmn085
255
2534
63plusmn10
2295
83378plusmn075
223
634
24plusmn088
258
634
21plusmn
089
259
3425plusmn093
272
103595plusmn11
2312
53526plusmn047
134
534
68plusmn00
63
180
63530plusmn074
209
3526plusmn090
256
53384plusmn062
184
3mdash
mdash0
mdashmdash
034
83plusmn060
171
3384plusmn062
184
QuantiTectR
T+qP
CRQuantitect
250
3137plusmn069
220
83145plusmn052
165
83122plusmn046
147
83135plusmn056
177
3135plusmn055
175
100
3246plusmn080
246
73272plusmn092
281
73390plusmn090
265
73303plusmn087
264
3303plusmn10
5318
503359plusmn060
179
43434plusmn232
675
53366plusmn079
235
63386plusmn12
4363
3387plusmn14
0413
253298plusmn074
224
43358plusmn118
351
53412plusmn207
608
43356plusmn13
3394
3356plusmn13
8410
103354plusmn018
054
33386plusmn061
180
23473plusmn062
179
23404plusmn047
138
3397plusmn065
192
5mdash
mdash0
3552
mdash1
mdashmdash
03552
mdash3552
mdash50
034
09plusmn036
135
836
37plusmn118
326
63297plusmn038
116
834
30plusmn15
419
234
30plusmn15
4448
250
3642plusmn046
643
83366plusmn080
239
83361plusmn
083
246
834
56plusmn19
7376
3456plusmn19
7569
100
3439plusmn234
257
834
60plusmn098
284
834
50plusmn091
265
834
50plusmn089
269
3450plusmn089
258
5036
28plusmn088
475
63507plusmn091
259
734
92plusmn10
4297
73538plusmn13
2344
3538plusmn13
2374
253597plusmn17
2230
536
28plusmn088
242
436
44plusmn078
214
536
23plusmn079
229
3623plusmn079
217
10mdash
mdash0
mdashmdash
03765plusmn079
210
23765plusmn079
210
3765plusmn079
210
BioMed Research International 11
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
Maxim
aRT
+qP
CRMaxim
a
100
3183plusmn084
263
3138plusmn022
070
3009plusmn041
137
311plusmn049
157
311plusmn092
295
503335plusmn10
9326
83342plusmn088
264
53112plusmn064
205
83263plusmn087
265
3251plusmn
140
432
253367plusmn088
261
53277plusmn112
340
63247plusmn072
221
73297plusmn090
274
3290plusmn099
302
103716plusmn464
1249
33400plusmn14
1416
43435plusmn13
3388
53517plusmn246
684
3494plusmn263
754
53544plusmn024
068
23865
mdash1
3458plusmn12
4357
33622plusmn074
212
3555plusmn17
6496
25
mdashmdash
03314
mdash1
3347plusmn17
6526
23330plusmn17
6526
3336plusmn12
6377
250
2928plusmn058
199
829
05plusmn038
130
829
04plusmn039
135
829
12plusmn045
155
2912plusmn045
156
100
3053plusmn056
185
830
17plusmn048
159
830
31plusmn
043
143
830
34plusmn050
162
3034plusmn050
164
503154plusmn12
5397
83188plusmn13
7429
83172plusmn056
177
83171plusmn
108
334
3171plusmn
108
340
253300plusmn115
348
63238plusmn079
243
73238plusmn082
254
73256plusmn092
282
3256plusmn092
281
103308plusmn064
193
33302plusmn13
8417
43340plusmn14
6438
43317plusmn115
349
3317plusmn11
5347
503303plusmn015
045
23355
mdash1
3299
mdash1
3315plusmn028
045
3315plusmn028
085
qScriptR
T+qP
CRPerfe
cta
250
2704plusmn024
088
82675plusmn026
098
82677plusmn024
090
82685plusmn025
092
2685plusmn027
101
100
3054plusmn054
176
82984plusmn054
182
83046plusmn10
7350
83028plusmn072
236
3028plusmn079
261
503203plusmn290
905
73072plusmn089
292
83078plusmn082
267
83118plusmn15
4488
3114plusmn17
7568
253250plusmn098
302
73241plusmn
133
410
73363plusmn216
642
53285plusmn14
9452
3276plusmn14
9456
103222plusmn081
252
53287plusmn046
139
33270plusmn056
171
23260plusmn061
187
3251plusmn
069
211
53278plusmn015
045
2mdash
mdash0
3356plusmn15
5461
23318plusmn085
253
3318plusmn10
0303
250
2947plusmn022
074
829
49plusmn054
183
729
82plusmn038
129
829
60plusmn041
129
2960plusmn041
139
100
3099plusmn10
9352
83114plusmn12
6404
83102plusmn052
169
83105plusmn096
308
3105plusmn096
311
503187plusmn060
188
83137plusmn057
180
83313plusmn11
2337
83212plusmn10
8235
3212plusmn10
8335
253335plusmn299
896
73249plusmn068
211
83271plusmn
087
265
83283plusmn17
2457
3283plusmn17
2524
103300plusmn049
147
53345plusmn018
053
23298plusmn074
225
43307plusmn054
142
3307plusmn054
165
53304plusmn10
1306
23303plusmn074
223
23359
mdash1
3314plusmn067
265
3314plusmn067
203
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 7
and showed a detection of at least 10 copies120583L All RT-qPCRassays showed satisfactory efficiency varying between 93 and107 with detection of at least 100 copies120583LThe 119910-interceptvaried between 3801 and 4183 for all tested kits
32 Differences in RNA Detection between the Five Kits TestedUsing the One-Step Method Table 3 details the results of thecomparison of the five one-step kits
(i) Rotor Gene Q MDx Three optimised qRT-PCR assaysrevealed poor sensitivity and enabled detection of at least103 copies120583L Of the five kits tested two kits (QuantiTect-1 step and Verso-1 step) yielded greater sensitivity with thedetection of at least 10 copies120583L (3 positive samples out of3 tested) All kits showed a satisfactory efficiency varyingbetween 94 and 104The119910-intercept varied between 3278and 3767 for the five tested one-step kits
A high presence of Primer-Dimer was shown for lowRNA concentrations for three kits SSIII Platinium-1 stepVerso-1 step and qScript-1 step
(ii) Mx3005P Of the five one-step kits tested using theMx3005P cycler three optimised one-step kits (ie Quanti-tect-1 step Rotor Gene-1 step and SSIII Platinium-1 step)revealed good sensitivity with the detection of at least10 copies120583L The two other optimised one-step assays (ieVerso-1 step and qScript-1 step) revealed poor sensitivitywith a detection of 102 and 103 copies120583L respectively Theamplification efficiencywas satisfactory for all kits (94ndash100)except for the qScript one-step kit (efficiency of 87) The 119910-intercept varied between 3680 and 4074 for all tested kitsexcept for the qScript kit (Intercept of 4074)
33 Comparison of Assay Performance
331 Limit of Detection at 95 for One-Step and Two-StepMethods The results of LOD 95 for the ten kits tested usingtwo-step and one-step methods are recorded in Table 4
(i) Rotor Gene Q MDx The results showed an LOD 95 fortwo-step and one-step kits ranging from 41 to 171 copies120583Land from 20 to 497 copies120583L respectively (Table 4) Ofthe five commercial master mixes tested using the two-stepqPCR the RT SuperScript Vilo in combination with theExpress kit yielded greater sensitivity with an LOD 95 of41 copies120583L Maxima in combination with the RT Maximaand Perfecta in combination with the RT qScript rankedsecond and third with detection of 78 and 88 copies120583Lrespectively followed by the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 110 and 171 copies120583L respectively
In contrast the one-step qRT-PCR kit Quantitect-1 stepyielded the best levels of detection with an LOD 95 of20 copies120583L The four other tested one-step kits yieldedpoor detection with an LOD 95 varying between 127 and497 copies120583L
(ii) Mx3005P The LOD 95 for one-step and two-step kitsranged from 15 to 981 copies120583L and 45 to 160 copies120583L
respectivelyOf the five commercialmastermixes tested usingthe two-step qPCR the RT SuperScript Vilo in combinationwith the Express kit yielded greater sensitivity with an LOD95 of 45 copies120583L Perfecta in combination with qScriptRT and Maxima in combination with RT Maxima rankedsecond and third with a detection of 67 and 76 copies120583Lrespectively followed by the the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 88 and 160 copies120583L respectively
Of the five tested one-step kits two kits (ie Rotorgene-1 step and Quantitect-1 step) yielded the best levelsof detection with an LOD 95 of 15 and 22 copies120583Lrespectively SSIII Platinium-1 step ranked third with an LOD95 of 50 copies120583L followed by Verso-1 step (LOD 95 of101 copies120583L) and qScript-1 step (981 copies120583L)
332 Reproducibility of One-Step and Two-Step MethodsTable 5 shows the analysis results of reproducibility of one-step and two-step assays
(i) Rotor Gene Q MDx For all optimised one-step or two-step PCR assays the interassay CVs were lower than 10demonstrating good reproducibility for all tested kits [23]The interassay CVs for the two-step kits and the one-step kitsranged from 174 to 295 and 135 to 427 respectively whilethe intra-assay CVs ranged from 135 to 35 and 079 to 672for two-step and one-step kits respectively (Tables 5(a) and5(b)) In analysing the intra-assay CVs a somewhat similarvariability was shown for two-step and one-step reactions
The interassay CVs for the most efficient two-step kit(ie SuperScript Vilo in combination with qPCR Express)and the most efficient one-step kit (ie Quantitect-1 step)ranged from 195 to 336 respectively while the intra-assayCVs ranged from 185 to 211 and 079 to 216 for two-step andone-step kits respectively (Tables 5(a) and 5(b))
(ii) Mx3005P For all optimised one-step or two-step PCRassays the interassay CVs were lower than 10 demonstrat-ing good reproducibility for all tested kitsThe interassay CVsfor the two-step and one-step kits ranged from 214 to 33and 084 to 45 respectively while the intra-assay CVs rangedfrom 123 to 422 and 038 to 47 for two-step and one-stepkits respectively
34 Practicability of the Different Assays Costs and TimeTable 6 shows a practicability assessment of the differentreal-time assays including a comparison of cost pipettingsteps and thermocycling times Total reaction times for theten real-time assays ranged from 2h42 to 3h08 for two-stepkits and 2h18 to 2h57 for one-step kits with the one-stepSSIII Platinium kit requiring the shortest time and two-stepMaxima kit the longest regardless the machine used Thekits were ranked from 1 to 10 based on the cost per PCRreaction in increasing order from C108 (least expensive =1) to C1225 (most expensive = 10) Overall of the 10 mastermix kits tested the one-step kits were cheaper than the two-step kits (Table 6) For the one-step kits qScript was theleast costly followed by the Rotor Gene-1 step Verso-1 stepSSIII Platinium-1 step and the Quantitect-1 step which was
8 BioMed Research International
Table3Com
paris
onof
qRT-PC
RSY
BRGreen
(one-step)
kits
Results
offived
ifferentq
RT-PCR
kitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1in
vitro
RNAtranscrip
tsTestingof
different
qRT-PC
Rkits(one-step)
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Quantitect-1ste
pRo
torG
ene-1step
Verso-1step
SSIIIP
latinium-1ste
pqScript-1
step
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
2010
61666plusmn008
045
1715plusmn005
029
1699plusmn006
038
1452plusmn019
032
1347plusmn003
019
1779plusmn024
132
1588plusmn022
136
1834plusmn032
172
1622plusmn004
022
1848plusmn004
020
105
2010
52006plusmn010
052
2062plusmn004
017
2040plusmn011
052
1790plusmn021
118
1655plusmn003
019
2113plusmn045
212
1942plusmn020
104
2160plusmn015
071
1964plusmn041
211
2238plusmn015
068
104
2010
42358plusmn014
058
2391plusmn
008
033
2382plusmn020
083
2143plusmn012
057
1986plusmn004
018
2460plusmn020
083
2287plusmn016
069
2517plusmn017
069
2308plusmn027
116
2611plusmn010
038
103
2010
32670plusmn024
091
2745plusmn028
101
2693plusmn003
011
2490plusmn023
092
2313plusmn016
067
2795plusmn026
093
2634plusmn033
124
2863plusmn030
106
2614plusmn010
038
2954plusmn025
086
102
2010
23042plusmn008
026
23lowastlowast
3019plusmn060
198
03lowastlowast
03lowastlowast
3198plusmn017
053
2977plusmn039
132
3174plusmn079
250
2893plusmn008
028
3107plusmn037
118
101
2010
13324plusmn029
087
13lowastlowast
3327plusmn15
3460
03lowastlowast
03lowastlowast
3434plusmn10
3299
3284plusmn044
133
23lowastlowast
2991plusmn
010
033
03lowastlowast
12
03lowastlowast
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
03lowastlowast
03lowastlowast
Efficiency
98
96
100
94
104
97
94
95
100
87
1198772
0998
0999
0999
0998
0999
099
6099
8099
7099
6099
8
119910-in
tercept
3689
3767
3699
3529
3278
3815
3680
3893
3893
4074
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 9
Table4Detectio
nlim
itof
PCR(LOD95)for
two-ste
pandon
e-ste
pSY
BRGreen
RT-PCR
Two-ste
pRT
-qPC
RIScriptR
T+qP
CRSsoF
ast
BioR
adQuantiTectR
T+qP
CRQuantitect
QIAGEN
Maxim
aRT+qP
CRMaxim
aFE
RMEN
TAS
SSViloRT
+qP
CREx
press
INVITRO
GEN
qScriptR
T+qP
CRPerfe
cta
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
11055
171
8678
3941
2188
4488
44160
8076
3845
2267
34One-stepqR
T-PC
RQuantitect-1ste
pQIAGEN
RotorG
ene-1step
QIAGEN
Verso-1step
FERM
ENTA
SSSIIIP
latinium-1ste
pIN
VITRO
GEN
qScript-1
step
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
2040
497
994
127
254
321
641
406
812
2244
1530
101
202
50100
981
1962
lowastAllLO
D95results
werec
arrie
dou
tusin
gProb
itmod
ellowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
10 BioMed Research International
Table5Analysis
ofreprod
ucibilityof
one-ste
pandtwo-ste
pRT
-PCR
(a)
show
sfor
each
two-ste
pkittheintra-a
ndinterassay
reprod
ucibilityforsixdilutio
nsof
EBLV
-1teste
deighttim
esin
each
ofthreeind
ependent
runs
(119899=144samples)a
nd(b)sho
wsthe
inter-andintra-assayreprod
ucibilityfora
llteste
don
e-ste
pkits
(a)2-ste
pmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
IScriptR
T+qP
CRSsoF
ast
100
3046plusmn041
135
83008plusmn057
190
82982plusmn041
138
83012plusmn046
154
3012plusmn052
174
503111plusmn093
299
63184plusmn10
1319
63080plusmn046
151
83125plusmn080
256
3120plusmn088
283
253249plusmn059
183
63160plusmn067
212
83127plusmn068
219
63179plusmn065
204
3177plusmn080
251
103238plusmn091
281
63269plusmn119
363
63197plusmn076
239
53235plusmn095
294
3237plusmn097
299
53314
mdash1
mdashmdash
03198plusmn005
015
33256plusmn005
015
3227plusmn058
181
05
3217
mdash1
mdashmdash
03211plusmn076
236
33214plusmn076
236
3213plusmn062
193
250
3071plusmn
049
161
830
70plusmn039
128
830
61plusmn
033
108
830
67plusmn041
132
3067plusmn040
129
100
3230plusmn074
228
83203plusmn090
281
83219plusmn040
124
83217plusmn068
211
3217plusmn069
214
5034
02plusmn093
273
63324plusmn048
144
83316plusmn093
287
83347plusmn079
235
3342plusmn085
255
2534
63plusmn10
2295
83378plusmn075
223
634
24plusmn088
258
634
21plusmn
089
259
3425plusmn093
272
103595plusmn11
2312
53526plusmn047
134
534
68plusmn00
63
180
63530plusmn074
209
3526plusmn090
256
53384plusmn062
184
3mdash
mdash0
mdashmdash
034
83plusmn060
171
3384plusmn062
184
QuantiTectR
T+qP
CRQuantitect
250
3137plusmn069
220
83145plusmn052
165
83122plusmn046
147
83135plusmn056
177
3135plusmn055
175
100
3246plusmn080
246
73272plusmn092
281
73390plusmn090
265
73303plusmn087
264
3303plusmn10
5318
503359plusmn060
179
43434plusmn232
675
53366plusmn079
235
63386plusmn12
4363
3387plusmn14
0413
253298plusmn074
224
43358plusmn118
351
53412plusmn207
608
43356plusmn13
3394
3356plusmn13
8410
103354plusmn018
054
33386plusmn061
180
23473plusmn062
179
23404plusmn047
138
3397plusmn065
192
5mdash
mdash0
3552
mdash1
mdashmdash
03552
mdash3552
mdash50
034
09plusmn036
135
836
37plusmn118
326
63297plusmn038
116
834
30plusmn15
419
234
30plusmn15
4448
250
3642plusmn046
643
83366plusmn080
239
83361plusmn
083
246
834
56plusmn19
7376
3456plusmn19
7569
100
3439plusmn234
257
834
60plusmn098
284
834
50plusmn091
265
834
50plusmn089
269
3450plusmn089
258
5036
28plusmn088
475
63507plusmn091
259
734
92plusmn10
4297
73538plusmn13
2344
3538plusmn13
2374
253597plusmn17
2230
536
28plusmn088
242
436
44plusmn078
214
536
23plusmn079
229
3623plusmn079
217
10mdash
mdash0
mdashmdash
03765plusmn079
210
23765plusmn079
210
3765plusmn079
210
BioMed Research International 11
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
Maxim
aRT
+qP
CRMaxim
a
100
3183plusmn084
263
3138plusmn022
070
3009plusmn041
137
311plusmn049
157
311plusmn092
295
503335plusmn10
9326
83342plusmn088
264
53112plusmn064
205
83263plusmn087
265
3251plusmn
140
432
253367plusmn088
261
53277plusmn112
340
63247plusmn072
221
73297plusmn090
274
3290plusmn099
302
103716plusmn464
1249
33400plusmn14
1416
43435plusmn13
3388
53517plusmn246
684
3494plusmn263
754
53544plusmn024
068
23865
mdash1
3458plusmn12
4357
33622plusmn074
212
3555plusmn17
6496
25
mdashmdash
03314
mdash1
3347plusmn17
6526
23330plusmn17
6526
3336plusmn12
6377
250
2928plusmn058
199
829
05plusmn038
130
829
04plusmn039
135
829
12plusmn045
155
2912plusmn045
156
100
3053plusmn056
185
830
17plusmn048
159
830
31plusmn
043
143
830
34plusmn050
162
3034plusmn050
164
503154plusmn12
5397
83188plusmn13
7429
83172plusmn056
177
83171plusmn
108
334
3171plusmn
108
340
253300plusmn115
348
63238plusmn079
243
73238plusmn082
254
73256plusmn092
282
3256plusmn092
281
103308plusmn064
193
33302plusmn13
8417
43340plusmn14
6438
43317plusmn115
349
3317plusmn11
5347
503303plusmn015
045
23355
mdash1
3299
mdash1
3315plusmn028
045
3315plusmn028
085
qScriptR
T+qP
CRPerfe
cta
250
2704plusmn024
088
82675plusmn026
098
82677plusmn024
090
82685plusmn025
092
2685plusmn027
101
100
3054plusmn054
176
82984plusmn054
182
83046plusmn10
7350
83028plusmn072
236
3028plusmn079
261
503203plusmn290
905
73072plusmn089
292
83078plusmn082
267
83118plusmn15
4488
3114plusmn17
7568
253250plusmn098
302
73241plusmn
133
410
73363plusmn216
642
53285plusmn14
9452
3276plusmn14
9456
103222plusmn081
252
53287plusmn046
139
33270plusmn056
171
23260plusmn061
187
3251plusmn
069
211
53278plusmn015
045
2mdash
mdash0
3356plusmn15
5461
23318plusmn085
253
3318plusmn10
0303
250
2947plusmn022
074
829
49plusmn054
183
729
82plusmn038
129
829
60plusmn041
129
2960plusmn041
139
100
3099plusmn10
9352
83114plusmn12
6404
83102plusmn052
169
83105plusmn096
308
3105plusmn096
311
503187plusmn060
188
83137plusmn057
180
83313plusmn11
2337
83212plusmn10
8235
3212plusmn10
8335
253335plusmn299
896
73249plusmn068
211
83271plusmn
087
265
83283plusmn17
2457
3283plusmn17
2524
103300plusmn049
147
53345plusmn018
053
23298plusmn074
225
43307plusmn054
142
3307plusmn054
165
53304plusmn10
1306
23303plusmn074
223
23359
mdash1
3314plusmn067
265
3314plusmn067
203
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
8 BioMed Research International
Table3Com
paris
onof
qRT-PC
RSY
BRGreen
(one-step)
kits
Results
offived
ifferentq
RT-PCR
kitson
arange
ofseventriplicated
ilutio
nsof
EBLV
-1in
vitro
RNAtranscrip
tsTestingof
different
qRT-PC
Rkits(one-step)
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Quantitect-1ste
pRo
torG
ene-1step
Verso-1step
SSIIIP
latinium-1ste
pqScript-1
step
AverageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowastAv
erageC
tlowastlowastlowast
CV(
)lowast
106
2010
61666plusmn008
045
1715plusmn005
029
1699plusmn006
038
1452plusmn019
032
1347plusmn003
019
1779plusmn024
132
1588plusmn022
136
1834plusmn032
172
1622plusmn004
022
1848plusmn004
020
105
2010
52006plusmn010
052
2062plusmn004
017
2040plusmn011
052
1790plusmn021
118
1655plusmn003
019
2113plusmn045
212
1942plusmn020
104
2160plusmn015
071
1964plusmn041
211
2238plusmn015
068
104
2010
42358plusmn014
058
2391plusmn
008
033
2382plusmn020
083
2143plusmn012
057
1986plusmn004
018
2460plusmn020
083
2287plusmn016
069
2517plusmn017
069
2308plusmn027
116
2611plusmn010
038
103
2010
32670plusmn024
091
2745plusmn028
101
2693plusmn003
011
2490plusmn023
092
2313plusmn016
067
2795plusmn026
093
2634plusmn033
124
2863plusmn030
106
2614plusmn010
038
2954plusmn025
086
102
2010
23042plusmn008
026
23lowastlowast
3019plusmn060
198
03lowastlowast
03lowastlowast
3198plusmn017
053
2977plusmn039
132
3174plusmn079
250
2893plusmn008
028
3107plusmn037
118
101
2010
13324plusmn029
087
13lowastlowast
3327plusmn15
3460
03lowastlowast
03lowastlowast
3434plusmn10
3299
3284plusmn044
133
23lowastlowast
2991plusmn
010
033
03lowastlowast
12
03lowastlowast
13lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
03lowastlowast
23lowastlowast
03lowastlowast
03lowastlowast
Efficiency
98
96
100
94
104
97
94
95
100
87
1198772
0998
0999
0999
0998
0999
099
6099
8099
7099
6099
8
119910-in
tercept
3689
3767
3699
3529
3278
3815
3680
3893
3893
4074
lowastCV
()=
standard
deviationmeanCttimes100
CVwas
notcalculated
whensomeo
fthe
parallelsfailedto
amplify
lowastlowast119883119883num
bero
fpositivesnu
mbero
fexp
erim
ents
lowastlowastlowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 9
Table4Detectio
nlim
itof
PCR(LOD95)for
two-ste
pandon
e-ste
pSY
BRGreen
RT-PCR
Two-ste
pRT
-qPC
RIScriptR
T+qP
CRSsoF
ast
BioR
adQuantiTectR
T+qP
CRQuantitect
QIAGEN
Maxim
aRT+qP
CRMaxim
aFE
RMEN
TAS
SSViloRT
+qP
CREx
press
INVITRO
GEN
qScriptR
T+qP
CRPerfe
cta
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
11055
171
8678
3941
2188
4488
44160
8076
3845
2267
34One-stepqR
T-PC
RQuantitect-1ste
pQIAGEN
RotorG
ene-1step
QIAGEN
Verso-1step
FERM
ENTA
SSSIIIP
latinium-1ste
pIN
VITRO
GEN
qScript-1
step
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
2040
497
994
127
254
321
641
406
812
2244
1530
101
202
50100
981
1962
lowastAllLO
D95results
werec
arrie
dou
tusin
gProb
itmod
ellowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
10 BioMed Research International
Table5Analysis
ofreprod
ucibilityof
one-ste
pandtwo-ste
pRT
-PCR
(a)
show
sfor
each
two-ste
pkittheintra-a
ndinterassay
reprod
ucibilityforsixdilutio
nsof
EBLV
-1teste
deighttim
esin
each
ofthreeind
ependent
runs
(119899=144samples)a
nd(b)sho
wsthe
inter-andintra-assayreprod
ucibilityfora
llteste
don
e-ste
pkits
(a)2-ste
pmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
IScriptR
T+qP
CRSsoF
ast
100
3046plusmn041
135
83008plusmn057
190
82982plusmn041
138
83012plusmn046
154
3012plusmn052
174
503111plusmn093
299
63184plusmn10
1319
63080plusmn046
151
83125plusmn080
256
3120plusmn088
283
253249plusmn059
183
63160plusmn067
212
83127plusmn068
219
63179plusmn065
204
3177plusmn080
251
103238plusmn091
281
63269plusmn119
363
63197plusmn076
239
53235plusmn095
294
3237plusmn097
299
53314
mdash1
mdashmdash
03198plusmn005
015
33256plusmn005
015
3227plusmn058
181
05
3217
mdash1
mdashmdash
03211plusmn076
236
33214plusmn076
236
3213plusmn062
193
250
3071plusmn
049
161
830
70plusmn039
128
830
61plusmn
033
108
830
67plusmn041
132
3067plusmn040
129
100
3230plusmn074
228
83203plusmn090
281
83219plusmn040
124
83217plusmn068
211
3217plusmn069
214
5034
02plusmn093
273
63324plusmn048
144
83316plusmn093
287
83347plusmn079
235
3342plusmn085
255
2534
63plusmn10
2295
83378plusmn075
223
634
24plusmn088
258
634
21plusmn
089
259
3425plusmn093
272
103595plusmn11
2312
53526plusmn047
134
534
68plusmn00
63
180
63530plusmn074
209
3526plusmn090
256
53384plusmn062
184
3mdash
mdash0
mdashmdash
034
83plusmn060
171
3384plusmn062
184
QuantiTectR
T+qP
CRQuantitect
250
3137plusmn069
220
83145plusmn052
165
83122plusmn046
147
83135plusmn056
177
3135plusmn055
175
100
3246plusmn080
246
73272plusmn092
281
73390plusmn090
265
73303plusmn087
264
3303plusmn10
5318
503359plusmn060
179
43434plusmn232
675
53366plusmn079
235
63386plusmn12
4363
3387plusmn14
0413
253298plusmn074
224
43358plusmn118
351
53412plusmn207
608
43356plusmn13
3394
3356plusmn13
8410
103354plusmn018
054
33386plusmn061
180
23473plusmn062
179
23404plusmn047
138
3397plusmn065
192
5mdash
mdash0
3552
mdash1
mdashmdash
03552
mdash3552
mdash50
034
09plusmn036
135
836
37plusmn118
326
63297plusmn038
116
834
30plusmn15
419
234
30plusmn15
4448
250
3642plusmn046
643
83366plusmn080
239
83361plusmn
083
246
834
56plusmn19
7376
3456plusmn19
7569
100
3439plusmn234
257
834
60plusmn098
284
834
50plusmn091
265
834
50plusmn089
269
3450plusmn089
258
5036
28plusmn088
475
63507plusmn091
259
734
92plusmn10
4297
73538plusmn13
2344
3538plusmn13
2374
253597plusmn17
2230
536
28plusmn088
242
436
44plusmn078
214
536
23plusmn079
229
3623plusmn079
217
10mdash
mdash0
mdashmdash
03765plusmn079
210
23765plusmn079
210
3765plusmn079
210
BioMed Research International 11
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
Maxim
aRT
+qP
CRMaxim
a
100
3183plusmn084
263
3138plusmn022
070
3009plusmn041
137
311plusmn049
157
311plusmn092
295
503335plusmn10
9326
83342plusmn088
264
53112plusmn064
205
83263plusmn087
265
3251plusmn
140
432
253367plusmn088
261
53277plusmn112
340
63247plusmn072
221
73297plusmn090
274
3290plusmn099
302
103716plusmn464
1249
33400plusmn14
1416
43435plusmn13
3388
53517plusmn246
684
3494plusmn263
754
53544plusmn024
068
23865
mdash1
3458plusmn12
4357
33622plusmn074
212
3555plusmn17
6496
25
mdashmdash
03314
mdash1
3347plusmn17
6526
23330plusmn17
6526
3336plusmn12
6377
250
2928plusmn058
199
829
05plusmn038
130
829
04plusmn039
135
829
12plusmn045
155
2912plusmn045
156
100
3053plusmn056
185
830
17plusmn048
159
830
31plusmn
043
143
830
34plusmn050
162
3034plusmn050
164
503154plusmn12
5397
83188plusmn13
7429
83172plusmn056
177
83171plusmn
108
334
3171plusmn
108
340
253300plusmn115
348
63238plusmn079
243
73238plusmn082
254
73256plusmn092
282
3256plusmn092
281
103308plusmn064
193
33302plusmn13
8417
43340plusmn14
6438
43317plusmn115
349
3317plusmn11
5347
503303plusmn015
045
23355
mdash1
3299
mdash1
3315plusmn028
045
3315plusmn028
085
qScriptR
T+qP
CRPerfe
cta
250
2704plusmn024
088
82675plusmn026
098
82677plusmn024
090
82685plusmn025
092
2685plusmn027
101
100
3054plusmn054
176
82984plusmn054
182
83046plusmn10
7350
83028plusmn072
236
3028plusmn079
261
503203plusmn290
905
73072plusmn089
292
83078plusmn082
267
83118plusmn15
4488
3114plusmn17
7568
253250plusmn098
302
73241plusmn
133
410
73363plusmn216
642
53285plusmn14
9452
3276plusmn14
9456
103222plusmn081
252
53287plusmn046
139
33270plusmn056
171
23260plusmn061
187
3251plusmn
069
211
53278plusmn015
045
2mdash
mdash0
3356plusmn15
5461
23318plusmn085
253
3318plusmn10
0303
250
2947plusmn022
074
829
49plusmn054
183
729
82plusmn038
129
829
60plusmn041
129
2960plusmn041
139
100
3099plusmn10
9352
83114plusmn12
6404
83102plusmn052
169
83105plusmn096
308
3105plusmn096
311
503187plusmn060
188
83137plusmn057
180
83313plusmn11
2337
83212plusmn10
8235
3212plusmn10
8335
253335plusmn299
896
73249plusmn068
211
83271plusmn
087
265
83283plusmn17
2457
3283plusmn17
2524
103300plusmn049
147
53345plusmn018
053
23298plusmn074
225
43307plusmn054
142
3307plusmn054
165
53304plusmn10
1306
23303plusmn074
223
23359
mdash1
3314plusmn067
265
3314plusmn067
203
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 9
Table4Detectio
nlim
itof
PCR(LOD95)for
two-ste
pandon
e-ste
pSY
BRGreen
RT-PCR
Two-ste
pRT
-qPC
RIScriptR
T+qP
CRSsoF
ast
BioR
adQuantiTectR
T+qP
CRQuantitect
QIAGEN
Maxim
aRT+qP
CRMaxim
aFE
RMEN
TAS
SSViloRT
+qP
CREx
press
INVITRO
GEN
qScriptR
T+qP
CRPerfe
cta
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
11055
171
8678
3941
2188
4488
44160
8076
3845
2267
34One-stepqR
T-PC
RQuantitect-1ste
pQIAGEN
RotorG
ene-1step
QIAGEN
Verso-1step
FERM
ENTA
SSSIIIP
latinium-1ste
pIN
VITRO
GEN
qScript-1
step
QUA
NTA
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
Num
bero
fcopiessam
ple
Num
bero
fcopiesrxn
2040
497
994
127
254
321
641
406
812
2244
1530
101
202
50100
981
1962
lowastAllLO
D95results
werec
arrie
dou
tusin
gProb
itmod
ellowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
10 BioMed Research International
Table5Analysis
ofreprod
ucibilityof
one-ste
pandtwo-ste
pRT
-PCR
(a)
show
sfor
each
two-ste
pkittheintra-a
ndinterassay
reprod
ucibilityforsixdilutio
nsof
EBLV
-1teste
deighttim
esin
each
ofthreeind
ependent
runs
(119899=144samples)a
nd(b)sho
wsthe
inter-andintra-assayreprod
ucibilityfora
llteste
don
e-ste
pkits
(a)2-ste
pmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
IScriptR
T+qP
CRSsoF
ast
100
3046plusmn041
135
83008plusmn057
190
82982plusmn041
138
83012plusmn046
154
3012plusmn052
174
503111plusmn093
299
63184plusmn10
1319
63080plusmn046
151
83125plusmn080
256
3120plusmn088
283
253249plusmn059
183
63160plusmn067
212
83127plusmn068
219
63179plusmn065
204
3177plusmn080
251
103238plusmn091
281
63269plusmn119
363
63197plusmn076
239
53235plusmn095
294
3237plusmn097
299
53314
mdash1
mdashmdash
03198plusmn005
015
33256plusmn005
015
3227plusmn058
181
05
3217
mdash1
mdashmdash
03211plusmn076
236
33214plusmn076
236
3213plusmn062
193
250
3071plusmn
049
161
830
70plusmn039
128
830
61plusmn
033
108
830
67plusmn041
132
3067plusmn040
129
100
3230plusmn074
228
83203plusmn090
281
83219plusmn040
124
83217plusmn068
211
3217plusmn069
214
5034
02plusmn093
273
63324plusmn048
144
83316plusmn093
287
83347plusmn079
235
3342plusmn085
255
2534
63plusmn10
2295
83378plusmn075
223
634
24plusmn088
258
634
21plusmn
089
259
3425plusmn093
272
103595plusmn11
2312
53526plusmn047
134
534
68plusmn00
63
180
63530plusmn074
209
3526plusmn090
256
53384plusmn062
184
3mdash
mdash0
mdashmdash
034
83plusmn060
171
3384plusmn062
184
QuantiTectR
T+qP
CRQuantitect
250
3137plusmn069
220
83145plusmn052
165
83122plusmn046
147
83135plusmn056
177
3135plusmn055
175
100
3246plusmn080
246
73272plusmn092
281
73390plusmn090
265
73303plusmn087
264
3303plusmn10
5318
503359plusmn060
179
43434plusmn232
675
53366plusmn079
235
63386plusmn12
4363
3387plusmn14
0413
253298plusmn074
224
43358plusmn118
351
53412plusmn207
608
43356plusmn13
3394
3356plusmn13
8410
103354plusmn018
054
33386plusmn061
180
23473plusmn062
179
23404plusmn047
138
3397plusmn065
192
5mdash
mdash0
3552
mdash1
mdashmdash
03552
mdash3552
mdash50
034
09plusmn036
135
836
37plusmn118
326
63297plusmn038
116
834
30plusmn15
419
234
30plusmn15
4448
250
3642plusmn046
643
83366plusmn080
239
83361plusmn
083
246
834
56plusmn19
7376
3456plusmn19
7569
100
3439plusmn234
257
834
60plusmn098
284
834
50plusmn091
265
834
50plusmn089
269
3450plusmn089
258
5036
28plusmn088
475
63507plusmn091
259
734
92plusmn10
4297
73538plusmn13
2344
3538plusmn13
2374
253597plusmn17
2230
536
28plusmn088
242
436
44plusmn078
214
536
23plusmn079
229
3623plusmn079
217
10mdash
mdash0
mdashmdash
03765plusmn079
210
23765plusmn079
210
3765plusmn079
210
BioMed Research International 11
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
Maxim
aRT
+qP
CRMaxim
a
100
3183plusmn084
263
3138plusmn022
070
3009plusmn041
137
311plusmn049
157
311plusmn092
295
503335plusmn10
9326
83342plusmn088
264
53112plusmn064
205
83263plusmn087
265
3251plusmn
140
432
253367plusmn088
261
53277plusmn112
340
63247plusmn072
221
73297plusmn090
274
3290plusmn099
302
103716plusmn464
1249
33400plusmn14
1416
43435plusmn13
3388
53517plusmn246
684
3494plusmn263
754
53544plusmn024
068
23865
mdash1
3458plusmn12
4357
33622plusmn074
212
3555plusmn17
6496
25
mdashmdash
03314
mdash1
3347plusmn17
6526
23330plusmn17
6526
3336plusmn12
6377
250
2928plusmn058
199
829
05plusmn038
130
829
04plusmn039
135
829
12plusmn045
155
2912plusmn045
156
100
3053plusmn056
185
830
17plusmn048
159
830
31plusmn
043
143
830
34plusmn050
162
3034plusmn050
164
503154plusmn12
5397
83188plusmn13
7429
83172plusmn056
177
83171plusmn
108
334
3171plusmn
108
340
253300plusmn115
348
63238plusmn079
243
73238plusmn082
254
73256plusmn092
282
3256plusmn092
281
103308plusmn064
193
33302plusmn13
8417
43340plusmn14
6438
43317plusmn115
349
3317plusmn11
5347
503303plusmn015
045
23355
mdash1
3299
mdash1
3315plusmn028
045
3315plusmn028
085
qScriptR
T+qP
CRPerfe
cta
250
2704plusmn024
088
82675plusmn026
098
82677plusmn024
090
82685plusmn025
092
2685plusmn027
101
100
3054plusmn054
176
82984plusmn054
182
83046plusmn10
7350
83028plusmn072
236
3028plusmn079
261
503203plusmn290
905
73072plusmn089
292
83078plusmn082
267
83118plusmn15
4488
3114plusmn17
7568
253250plusmn098
302
73241plusmn
133
410
73363plusmn216
642
53285plusmn14
9452
3276plusmn14
9456
103222plusmn081
252
53287plusmn046
139
33270plusmn056
171
23260plusmn061
187
3251plusmn
069
211
53278plusmn015
045
2mdash
mdash0
3356plusmn15
5461
23318plusmn085
253
3318plusmn10
0303
250
2947plusmn022
074
829
49plusmn054
183
729
82plusmn038
129
829
60plusmn041
129
2960plusmn041
139
100
3099plusmn10
9352
83114plusmn12
6404
83102plusmn052
169
83105plusmn096
308
3105plusmn096
311
503187plusmn060
188
83137plusmn057
180
83313plusmn11
2337
83212plusmn10
8235
3212plusmn10
8335
253335plusmn299
896
73249plusmn068
211
83271plusmn
087
265
83283plusmn17
2457
3283plusmn17
2524
103300plusmn049
147
53345plusmn018
053
23298plusmn074
225
43307plusmn054
142
3307plusmn054
165
53304plusmn10
1306
23303plusmn074
223
23359
mdash1
3314plusmn067
265
3314plusmn067
203
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
10 BioMed Research International
Table5Analysis
ofreprod
ucibilityof
one-ste
pandtwo-ste
pRT
-PCR
(a)
show
sfor
each
two-ste
pkittheintra-a
ndinterassay
reprod
ucibilityforsixdilutio
nsof
EBLV
-1teste
deighttim
esin
each
ofthreeind
ependent
runs
(119899=144samples)a
nd(b)sho
wsthe
inter-andintra-assayreprod
ucibilityfora
llteste
don
e-ste
pkits
(a)2-ste
pmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
IScriptR
T+qP
CRSsoF
ast
100
3046plusmn041
135
83008plusmn057
190
82982plusmn041
138
83012plusmn046
154
3012plusmn052
174
503111plusmn093
299
63184plusmn10
1319
63080plusmn046
151
83125plusmn080
256
3120plusmn088
283
253249plusmn059
183
63160plusmn067
212
83127plusmn068
219
63179plusmn065
204
3177plusmn080
251
103238plusmn091
281
63269plusmn119
363
63197plusmn076
239
53235plusmn095
294
3237plusmn097
299
53314
mdash1
mdashmdash
03198plusmn005
015
33256plusmn005
015
3227plusmn058
181
05
3217
mdash1
mdashmdash
03211plusmn076
236
33214plusmn076
236
3213plusmn062
193
250
3071plusmn
049
161
830
70plusmn039
128
830
61plusmn
033
108
830
67plusmn041
132
3067plusmn040
129
100
3230plusmn074
228
83203plusmn090
281
83219plusmn040
124
83217plusmn068
211
3217plusmn069
214
5034
02plusmn093
273
63324plusmn048
144
83316plusmn093
287
83347plusmn079
235
3342plusmn085
255
2534
63plusmn10
2295
83378plusmn075
223
634
24plusmn088
258
634
21plusmn
089
259
3425plusmn093
272
103595plusmn11
2312
53526plusmn047
134
534
68plusmn00
63
180
63530plusmn074
209
3526plusmn090
256
53384plusmn062
184
3mdash
mdash0
mdashmdash
034
83plusmn060
171
3384plusmn062
184
QuantiTectR
T+qP
CRQuantitect
250
3137plusmn069
220
83145plusmn052
165
83122plusmn046
147
83135plusmn056
177
3135plusmn055
175
100
3246plusmn080
246
73272plusmn092
281
73390plusmn090
265
73303plusmn087
264
3303plusmn10
5318
503359plusmn060
179
43434plusmn232
675
53366plusmn079
235
63386plusmn12
4363
3387plusmn14
0413
253298plusmn074
224
43358plusmn118
351
53412plusmn207
608
43356plusmn13
3394
3356plusmn13
8410
103354plusmn018
054
33386plusmn061
180
23473plusmn062
179
23404plusmn047
138
3397plusmn065
192
5mdash
mdash0
3552
mdash1
mdashmdash
03552
mdash3552
mdash50
034
09plusmn036
135
836
37plusmn118
326
63297plusmn038
116
834
30plusmn15
419
234
30plusmn15
4448
250
3642plusmn046
643
83366plusmn080
239
83361plusmn
083
246
834
56plusmn19
7376
3456plusmn19
7569
100
3439plusmn234
257
834
60plusmn098
284
834
50plusmn091
265
834
50plusmn089
269
3450plusmn089
258
5036
28plusmn088
475
63507plusmn091
259
734
92plusmn10
4297
73538plusmn13
2344
3538plusmn13
2374
253597plusmn17
2230
536
28plusmn088
242
436
44plusmn078
214
536
23plusmn079
229
3623plusmn079
217
10mdash
mdash0
mdashmdash
03765plusmn079
210
23765plusmn079
210
3765plusmn079
210
BioMed Research International 11
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
Maxim
aRT
+qP
CRMaxim
a
100
3183plusmn084
263
3138plusmn022
070
3009plusmn041
137
311plusmn049
157
311plusmn092
295
503335plusmn10
9326
83342plusmn088
264
53112plusmn064
205
83263plusmn087
265
3251plusmn
140
432
253367plusmn088
261
53277plusmn112
340
63247plusmn072
221
73297plusmn090
274
3290plusmn099
302
103716plusmn464
1249
33400plusmn14
1416
43435plusmn13
3388
53517plusmn246
684
3494plusmn263
754
53544plusmn024
068
23865
mdash1
3458plusmn12
4357
33622plusmn074
212
3555plusmn17
6496
25
mdashmdash
03314
mdash1
3347plusmn17
6526
23330plusmn17
6526
3336plusmn12
6377
250
2928plusmn058
199
829
05plusmn038
130
829
04plusmn039
135
829
12plusmn045
155
2912plusmn045
156
100
3053plusmn056
185
830
17plusmn048
159
830
31plusmn
043
143
830
34plusmn050
162
3034plusmn050
164
503154plusmn12
5397
83188plusmn13
7429
83172plusmn056
177
83171plusmn
108
334
3171plusmn
108
340
253300plusmn115
348
63238plusmn079
243
73238plusmn082
254
73256plusmn092
282
3256plusmn092
281
103308plusmn064
193
33302plusmn13
8417
43340plusmn14
6438
43317plusmn115
349
3317plusmn11
5347
503303plusmn015
045
23355
mdash1
3299
mdash1
3315plusmn028
045
3315plusmn028
085
qScriptR
T+qP
CRPerfe
cta
250
2704plusmn024
088
82675plusmn026
098
82677plusmn024
090
82685plusmn025
092
2685plusmn027
101
100
3054plusmn054
176
82984plusmn054
182
83046plusmn10
7350
83028plusmn072
236
3028plusmn079
261
503203plusmn290
905
73072plusmn089
292
83078plusmn082
267
83118plusmn15
4488
3114plusmn17
7568
253250plusmn098
302
73241plusmn
133
410
73363plusmn216
642
53285plusmn14
9452
3276plusmn14
9456
103222plusmn081
252
53287plusmn046
139
33270plusmn056
171
23260plusmn061
187
3251plusmn
069
211
53278plusmn015
045
2mdash
mdash0
3356plusmn15
5461
23318plusmn085
253
3318plusmn10
0303
250
2947plusmn022
074
829
49plusmn054
183
729
82plusmn038
129
829
60plusmn041
129
2960plusmn041
139
100
3099plusmn10
9352
83114plusmn12
6404
83102plusmn052
169
83105plusmn096
308
3105plusmn096
311
503187plusmn060
188
83137plusmn057
180
83313plusmn11
2337
83212plusmn10
8235
3212plusmn10
8335
253335plusmn299
896
73249plusmn068
211
83271plusmn
087
265
83283plusmn17
2457
3283plusmn17
2524
103300plusmn049
147
53345plusmn018
053
23298plusmn074
225
43307plusmn054
142
3307plusmn054
165
53304plusmn10
1306
23303plusmn074
223
23359
mdash1
3314plusmn067
265
3314plusmn067
203
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 11
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
Maxim
aRT
+qP
CRMaxim
a
100
3183plusmn084
263
3138plusmn022
070
3009plusmn041
137
311plusmn049
157
311plusmn092
295
503335plusmn10
9326
83342plusmn088
264
53112plusmn064
205
83263plusmn087
265
3251plusmn
140
432
253367plusmn088
261
53277plusmn112
340
63247plusmn072
221
73297plusmn090
274
3290plusmn099
302
103716plusmn464
1249
33400plusmn14
1416
43435plusmn13
3388
53517plusmn246
684
3494plusmn263
754
53544plusmn024
068
23865
mdash1
3458plusmn12
4357
33622plusmn074
212
3555plusmn17
6496
25
mdashmdash
03314
mdash1
3347plusmn17
6526
23330plusmn17
6526
3336plusmn12
6377
250
2928plusmn058
199
829
05plusmn038
130
829
04plusmn039
135
829
12plusmn045
155
2912plusmn045
156
100
3053plusmn056
185
830
17plusmn048
159
830
31plusmn
043
143
830
34plusmn050
162
3034plusmn050
164
503154plusmn12
5397
83188plusmn13
7429
83172plusmn056
177
83171plusmn
108
334
3171plusmn
108
340
253300plusmn115
348
63238plusmn079
243
73238plusmn082
254
73256plusmn092
282
3256plusmn092
281
103308plusmn064
193
33302plusmn13
8417
43340plusmn14
6438
43317plusmn115
349
3317plusmn11
5347
503303plusmn015
045
23355
mdash1
3299
mdash1
3315plusmn028
045
3315plusmn028
085
qScriptR
T+qP
CRPerfe
cta
250
2704plusmn024
088
82675plusmn026
098
82677plusmn024
090
82685plusmn025
092
2685plusmn027
101
100
3054plusmn054
176
82984plusmn054
182
83046plusmn10
7350
83028plusmn072
236
3028plusmn079
261
503203plusmn290
905
73072plusmn089
292
83078plusmn082
267
83118plusmn15
4488
3114plusmn17
7568
253250plusmn098
302
73241plusmn
133
410
73363plusmn216
642
53285plusmn14
9452
3276plusmn14
9456
103222plusmn081
252
53287plusmn046
139
33270plusmn056
171
23260plusmn061
187
3251plusmn
069
211
53278plusmn015
045
2mdash
mdash0
3356plusmn15
5461
23318plusmn085
253
3318plusmn10
0303
250
2947plusmn022
074
829
49plusmn054
183
729
82plusmn038
129
829
60plusmn041
129
2960plusmn041
139
100
3099plusmn10
9352
83114plusmn12
6404
83102plusmn052
169
83105plusmn096
308
3105plusmn096
311
503187plusmn060
188
83137plusmn057
180
83313plusmn11
2337
83212plusmn10
8235
3212plusmn10
8335
253335plusmn299
896
73249plusmn068
211
83271plusmn
087
265
83283plusmn17
2457
3283plusmn17
2524
103300plusmn049
147
53345plusmn018
053
23298plusmn074
225
43307plusmn054
142
3307plusmn054
165
53304plusmn10
1306
23303plusmn074
223
23359
mdash1
3314plusmn067
265
3314plusmn067
203
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
12 BioMed Research International
(a)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CV ()lowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CV ()lowastlowast
AverageC
tplusmnSD
CtCV
()lowastlowast
SSViloRT
+qP
CREx
press
100
2768plusmn059
213
82782plusmn055
197
82759plusmn029
106
82770plusmn048
172
2770plusmn048
174
502942plusmn062
211
82927plusmn054
185
82921plusmn
060
206
82930plusmn059
201
2930plusmn057
195
253038plusmn067
221
63022plusmn12
4411
82960plusmn048
162
83007plusmn080
264
3004plusmn090
300
103136plusmn049
158
73130plusmn071
227
73192plusmn078
245
63153plusmn066
210
3151plusmn
069
219
53149plusmn053
168
43163plusmn098
308
43164
mdash1
3159plusmn075
238
3157plusmn068
216
05
mdashmdash
0mdash
mdash0
3159
mdash1
3159
mdash3159
mdash100
3178plusmn053
167
830
13plusmn032
107
830
75plusmn065
211
830
89plusmn085
162
3089plusmn085
276
503296plusmn042
129
83134plusmn050
161
83149plusmn097
307
83193plusmn099
199
3193plusmn099
309
253372plusmn10
5310
73215plusmn098
306
83209plusmn022
069
63266plusmn113
228
3266plusmn11
3346
1034
44plusmn060
174
73361plusmn
046
135
53330plusmn043
129
63383plusmn071
146
3383plusmn071
209
53521plusmn
053
151
73264plusmn036
111
53299plusmn006
017
23397plusmn13
5093
3397plusmn13
5398
25
3956plusmn18
6471
334
35
13355plusmn057
169
336
24plusmn332
320
3624plusmn332
916
Theintra-andinterassay
varia
bilitya
ndcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinter-assay
CV=sta
ndarddeviationofallreplicates(meanCt-value
ofallreplicates)times
100intra-assayC
V=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
(b)1stepmetho
ds
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Quantitect1step
502976plusmn064
216
82894plusmn055
189
82927plusmn023
079
82932plusmn047
161
2932plusmn059
106
253090plusmn088
286
82984plusmn10
9365
83098plusmn076
245
83057plusmn091
299
3057plusmn10
3202
103172plusmn10
4326
73205plusmn10
6332
73230plusmn061
188
83203plusmn090
282
3204plusmn090
336
53334plusmn12
0360
63238plusmn074
228
73285plusmn084
255
73286plusmn092
281
3283plusmn096
281
25
3251plusmn
060
185
43390plusmn033
096
43445plusmn15
2442
53362plusmn082
241
3368plusmn12
7377
13338plusmn096
288
43203plusmn026
080
33292plusmn047
143
53277plusmn056
170
3285plusmn079
242
5030
92plusmn047
152
83123plusmn12
9040
83111plusmn13
2041
83108plusmn13
8043
3108plusmn043
138
253177plusmn073
229
83192plusmn14
9048
83191plusmn
093
030
83187plusmn15
7050
3187plusmn051
161
103260plusmn047
144
83281plusmn
061
187
53249plusmn065
199
83263plusmn058
177
3263plusmn057
175
53369plusmn085
253
73382plusmn291
098
53328plusmn250
083
63360plusmn265
089
3357plusmn086
257
25
3436plusmn073
214
53365plusmn18
8063
23327plusmn18
8062
33376plusmn19
6066
3380plusmn080
236
134
55plusmn17
7513
334
97plusmn117
041
53392plusmn341
116
134
48plusmn324
111
3445plusmn13
7397
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 13
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
RotorG
ene1
step
1000
2640plusmn045
169
82374plusmn040
167
82701plusmn
118
438
82572plusmn16
2258
2572plusmn16
2632
500
2749plusmn046
166
82429plusmn034
139
82742plusmn10
4378
82640plusmn16
6228
2640plusmn16
6628
250
2873plusmn076
265
82490plusmn049
196
82927plusmn060
204
72756plusmn209
222
2756plusmn209
758
100
3070plusmn021
067
32550
mdash1
3119plusmn066
211
23000plusmn224
139
3000plusmn224
746
503031
mdash1
mdashmdash
0mdash
mdash0
3031
mdash3031
mdash25
3112plusmn050
161
2mdash
mdash0
mdashmdash
03212plusmn050
161
3212plusmn050
161
5028
87plusmn099
341
829
25plusmn074
252
82797plusmn065
233
828
70plusmn079
276
2870plusmn094
329
2529
49plusmn10
1344
830
36plusmn070
229
828
57plusmn039
136
829
47plusmn070
236
2947plusmn10
3350
1030
29plusmn12
0398
830
61plusmn
144
470
828
76plusmn040
139
829
89plusmn10
1336
2989plusmn13
4450
530
82plusmn10
6343
63147plusmn13
0412
629
90plusmn040
135
830
73plusmn092
297
3065plusmn11
2366
25
3087plusmn114
368
63169plusmn057
181
529
10plusmn037
126
530
55plusmn069
225
3057plusmn13
1429
13122
mdash1
3153
mdash1
3201plusmn
044
137
53159plusmn044
137
3183plusmn048
151
Verso1step
500
2650plusmn018
068
82656plusmn014
053
82679plusmn023
085
82661plusmn
018
069
2661plusmn
022
082
250
2743plusmn018
065
82796plusmn027
098
82789plusmn036
127
82776plusmn027
097
2776plusmn036
130
100
2894plusmn050
172
82942plusmn053
180
82991plusmn
118
396
82943plusmn074
249
2943plusmn087
295
503049plusmn044
143
73138plusmn086
274
83129plusmn15
2487
83105plusmn094
301
3108plusmn10
9350
253282plusmn114
346
73290plusmn052
157
53250plusmn092
282
73274plusmn086
262
3272plusmn090
274
103342plusmn097
291
33522plusmn10
5299
23442
mdash1
3435plusmn10
1295
3419plusmn118
344
250
2939plusmn048
164
829
40plusmn023
079
829
10plusmn053
183
829
20plusmn042
142
2930plusmn044
150
100
3055plusmn025
080
830
32plusmn038
125
830
02plusmn024
082
630
29plusmn029
096
3032plusmn036
119
503116plusmn050
159
83110plusmn052
168
830
87plusmn042
135
73104plusmn048
154
3105plusmn048
153
253171plusmn
032
102
73161plusmn
042
134
83135plusmn058
184
83156plusmn044
140
3155plusmn046
147
103186plusmn054
169
73176plusmn029
091
73165plusmn064
201
43176plusmn049
154
3177plusmn046
145
53309plusmn098
297
53313
13343plusmn074
223
43322plusmn086
260
3326plusmn080
239
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
14 BioMed Research International
(b)Con
tinued
Kit
Num
bero
fcopiessam
ple
Intra-assayvaria
bilitylowastlowastlowast
Inter-assayvaria
bilitylowastlowastlowast
Run1
Run2
Run3
Average
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sAv
erageC
tplusmnSD
Ctlowast
CVlowastlowast
Num
ber
ofpo
sCtplusmnSD
Ctlowast
CVlowastlowast
AverageC
tplusmnSD
Ctlowast
CVlowastlowast
qScript1
step
1000
2324plusmn012
053
82297plusmn024
103
82321plusmn
009
037
82314plusmn015
064
2314plusmn020
086
750
2364plusmn021
088
82329plusmn008
035
82352plusmn028
121
82348plusmn019
081
2348plusmn025
106
500
2446plusmn032
132
82406plusmn024
098
82407plusmn029
122
82419plusmn028
117
2419plusmn033
137
250
2538plusmn027
107
82493plusmn026
105
82514plusmn034
137
82515plusmn029
116
2515plusmn034
135
100
2626plusmn031
116
82579plusmn024
093
72595plusmn047
180
52600plusmn034
130
2602plusmn038
146
50mdash
mdash0
mdashmdash
02645plusmn018
070
22645plusmn018
070
2645plusmn018
070
2500
2813plusmn083
294
82778plusmn045
160
826
32plusmn030
113
82741plusmn
052
189
2741plusmn
097
353
1000
2878plusmn009
033
829
40plusmn15
8539
824
89plusmn030
122
82769plusmn066
231
2769plusmn223
805
750
2908plusmn024
082
828
92plusmn025
085
828
75plusmn011
038
828
92plusmn020
068
2892plusmn024
084
500
3069plusmn048
157
729
76plusmn055
186
82763plusmn056
201
529
36plusmn053
181
2966plusmn12
6424
250
3122plusmn023
073
730
05plusmn037
124
8mdash
mdash0
3064plusmn030
099
3060plusmn067
220
100
mdashmdash
030
00
mdash1
mdashmdash
030
00
mdash30
00
mdash
SSIIIP
latinium
1step
1000
2479plusmn024
097
82381plusmn
021
089
82446plusmn015
060
82435plusmn020
082
2435plusmn046
188
750
2518plusmn024
095
82430plusmn012
051
82497plusmn010
042
82482plusmn016
063
2482plusmn041
167
500
2666plusmn082
307
82536plusmn023
092
82619plusmn024
092
82607plusmn043
164
2607plusmn073
282
250
2866plusmn086
301
82838plusmn19
1672
82897plusmn059
205
82867plusmn112
393
2867plusmn12
2427
100
2917plusmn053
180
82778plusmn049
175
82816plusmn053
189
72837plusmn052
182
2838plusmn078
276
503095plusmn043
139
22911plusmn067
231
42993plusmn111
370
33000plusmn074
247
2979plusmn10
3347
250
2627plusmn026
099
826
70plusmn027
102
826
67plusmn042
156
826
55plusmn037
119
2655plusmn037
139
100
2722plusmn068
248
828
02plusmn039
138
82774plusmn034
121
82766plusmn058
169
2766plusmn058
209
502756plusmn053
194
828
26plusmn041
147
828
33plusmn040
141
828
05plusmn056
161
2805plusmn056
200
252787plusmn069
247
829
34plusmn055
189
828
58plusmn041
145
828
59plusmn082
194
2859plusmn082
285
1028
75plusmn078
270
629
44plusmn055
186
529
08plusmn033
114
729
07plusmn060
190
2907plusmn060
207
528
82plusmn015
053
428
71plusmn
021
072
328
88plusmn032
112
428
81plusmn
023
079
2881plusmn
023
079
Theintra-a
ndinterassay
varia
bilityandcoeffi
ciento
fvariatio
nswerec
alculated
bytheformulasinterassayCV
=standard
deviationof
allreplicates(meanCt-value
ofallreplicates)times
100intra-assayCV
=mean
oftheC
Vso
fallruns
lowastAv
erageC
tvaluesmeanplusmnstandard
deviation
lowastlowastCV
()=
standard
deviationmeanCttimes100
lowastlowastlowastAllPC
Rcomparis
onsw
erer
unon
theR
otor
GeneQ
MDxthermocycler(in
italic
font)a
ndon
theM
x3005P
thermocycler(in
bold)
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 15
Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps
Ranking forcostPCRreactionlowast
Run duration(minutes) Number
ofpipettingsteps lowastlowast
RotorGene QMDx
Mx3005P
Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14
Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10
One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7
qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps
the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast
4 Discussion
The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the
concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]
The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers
The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance
In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
16 BioMed Research International
using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples
Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits
The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS
SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]
PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]
5 Conclusion
With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 17
Abbreviations
Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination
Conflict of Interests
The authors declare that they have no competing interests
Acknowledgments
The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions
References
[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp
[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001
[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013
[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013
[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009
[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009
[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011
[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004
[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009
[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA
influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004
[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010
[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011
[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006
[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011
[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012
[18] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005
[19] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011
[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013
[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011
[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009
[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004
[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
18 BioMed Research International
[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004
[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010
[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014
[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012
[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014
[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013
[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004
[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012
[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005
[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003
[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005
[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008
[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology