USDA Avian Influenza Virus Diagnostic Workshop Iowa State University, Ames, Iowa

Molecular Diagnostics Laboratory

Real-time RT-PCR Introduction The goal of this laboratory is to provide an overview of real-time RT-PCR (RRT-PCR) as a diagnostic tool for avian influenza and Newcastle disease virus. RNA isolation, the RRT-PCR reaction and results interpretation will be demonstrated. USDA AVPRO1510 and AVPRO1505 further describe the RRT-PCR procedures for the detection of avian influenza and Newcastle disease virus. RT-PCR is a rapid method for the detection of RNA, the viral genetic material of avian influenza virus (AIV) and Newcastle disease virus (NDV). Like most nucleic acid based detection methods it is very sensitive and highly specific. Some of the advantages of RRT-PCR over virus isolation for AIV are a relatively low cost per sample, results available in as little as three hours, reduced handling of potentially infectious material and scalability.

Real-time RT-PCR will be the focus of this lab as it has been more widely used than standard RTPCR for influenza detection. RRT-PCR is set-up almost identically to standard RT-PCR, except for the specialized tubes and the addition of a dye labeled probe (more detail on the last page of this lab handout). The technique utilizes a one step protocol with specific primers designed to amplify a portion of the genome that contains a target PCR sequence. Non-extendible fluorogenic hydrolysis/Taqman probes monitor the target PCR product formation at each cycle during the PCR reaction. The probes are labeled at the 5’ end with a reporter dye (e.g. FAM) and a quencher dye (e.g. blackhole quencher (BHQ-1)) at the 3’ end. The proximally located quencher dye absorbs the emission of the reporter dye as long as the probe is intact and not hybridized to the target. When the probe is hybridized to the target, the 5’ nuclease activity of Taq-polymerase will cause hydrolysis of the probe, separating the quencher from the reporter dye. This separation results in an increase in fluorescence emission of the reporter dye, which is detected spectrophomectrically and recorded. The amount of fluorescence recorded is proportional to the amount of target template in the samples.

Real-time RT-PCR Advantages • Fast- results in as little as 3 hours • Sensitive • Specific • Scalable • Cost • Reduces handling of potentially infectious material • Viable virus not needed • Can test many sample types

Real-time RT-PCR disadvantages • Expensive initial investment (equipment) • Probes must be stored and handled correctly • False positives

� Cross contamination � Cross reactions/non-specific detection

• False negatives � Inhibitory substances in the sample � Template modification/ degradation: RNA fragile

During this laboratory we will extract RNA with a silicon-nucleic acid binding column (Qiagen RNeasy kit) and will be performing the RRT-PCR test for the avian influenza virus Matrix gene. The procedures for the avian influenza and Newcastle disease virus are similar, except for the primers and probes used and the temperature cycling conditions used. Specifics of each test are provided in the detailed protocols in your note book. For laboratories interested in standard RT-PCR, it is recommended that the primers used with realtime RT-PCR test not be used due to the small product size. A procedure for a standard RT-PCR for avian influenza has been reported by Fouchier, et al. (Fouchier, R. A., T. M. Bestebroer, S. Herfst, L. Van Der Kemp, G. F. Rimmelzwaan and A. D. Osterhaus. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J Clin Microbiol 38:4096-101. 2000.) A copy of which is provided in your notebook. RNA Extraction • Isolates RNA from other materials in the sample • Concentrates the RNA (5-10 times) • Removes inhibitory substances • Removes substances that will degrade the RNA Each student will extract RNA from 2 samples and will with the other members of their group set-up and run RRT-PCR reactions for AIV matrix or H5. Materials needed • 2 test samples for each person (4-5 lab members per group) • 1.5ml microfuge tubes • Pipets and tips • 2 RNeasy columns per person • Vacuum manifold and tubing • RNeasy Kit components

– RLT – RPE buffer with ethanol added – RW1 buffer – Nuclease free water (elution buffer)

Notes: • All Procedures should be carried out in a biological safety cabinet or other primary containment device.

• Kit supplied buffers should be prepared as specified in the kit instructions (i.e. 10μl per 1ml of 2-mercapto-ethanol should be added to the RLT buffer immediately prior to use).

• Wear gloves at all times during this procedure. RNA Extraction with Qiagen RNeasy Kit- QiaVac 24 Vacuum Manifold Method Procedure: 1. Each participant will start with two 1.5 ml centrifuge tube which will contain 500 µl of swab specimen. 2. Add to each microfuge tube from step 1

500μl RLT buffer 3. Vortex 15 sec. and pulse centrifuge. 4. Add 500μl 70% ethanol to the lysed swab specimen. Vortex for 15 sec. Centrifuge for 5 minutes at 5000 Xg at RT. 5. Set-up the vacuum manifold: place the appropriate number of RNeasy columns in the luer locks of the vacuum manifold, cover any empty positions with the luer caps supplied with the vacuum manifold. 6. Apply vacuum and add the entire sample/RLT/ethanol mixture to an RNeasy column for each sample. Press down on the top of the manifold once the vacuum is on to seal the manifold, then open the lids of all the columns and keep them open at all times. The vacuum should not be turned off until after the final wash. 7. Wash by applying 700μl RW1 buffer to each column. 8. Wash again by applying 500μl RPE buffer to the column and repeat for a total of 2 washes with buffer RPE. 9. Shut off the vacuum and place each RNeasy column in a 2ml collection tube. 10. Centrifuge the column and collection tube for 2 minutes at ~14 KXg and discard the collection tube. 11. Place the column in an elution tube (or 1.5ml microfuge tube)

Steps 11-13 place in elution tube, add water and elute RNA by centrifugation

Steps 9-10 put column in tube and centrifuge todry

Steps 1 - 5 in a 1.5ml tube

Step 6 -8 - turn on vacuum and add sample to column and wash

12. Add 50μl nuclease free water to the column and incubate at room temperature 1 minute. 13. Elute RNA by centrifuging for 1 minute at ~14KXg. Store at -70°C long term. Real-time RT-PCR • RT- reverse transcription

o cDNA is made from the viral RNA � Adds time to test

• PCR- polymerase chain reaction o cDNA is amplified o DNA is more stable and more easily amplified than RNA

• Real-time o Increases in the amount of DNA produced are detected as they occur

Each lab-group will need: – Smart Cycler tube cooling block – Smart Cycler tubes – A set of RT-PCR reagents

• Enzyme and RNase inhibitor in a bench top cooler • 5X buffer • dNTPS • Positive control (AIV M or H5 transcribed RNA positive control) • Nuclease free water • Forward primer (Matrix or H5) • Reverse primer (Matrix or H5) • Probe (Matrix or H5)

– RNA samples – Pipets and aerosol resistant pipet tips – 1.5ml tubes Real-time RT-PCR for type A Influenza (MA gene) Procedure: • Wear gloves at all times during this procedure. • This should be performed in a biological safety cabinet or similar device The Smart Cycler has already been programmed to run the sample with the conditions given in tables 1 and 2.

Table 1. RT step thermocycling for Qiagen one-step RT-PCR Kit. RT Step 1 cycle 30 min. 50 C 15 min. 95 C

Table 2. Thermocycling conditions for gene specific probe and primer sets. Probe/Primer set Step Time Temp Type A influenza 45 cycles denaturation 1 sec. 94 C (MA gene) Annealing* 20 sec. 60 C Subtype H5 (HA gene) 40 cycles Denaturation 1 sec. 94 C Annealing* 20 sec. 57 C Extension 5 sec. 72 C *Note: The fluorescence is acquired at the annealing step. 1. In a new, clean 1.5 ml tube prepare the reaction master mix (everything but the template) as shown in table. 3. Add the enzyme and probe last. Step 1 Notes: • The probe is light sensitive and when working in a biological safely cabinet the light should be turned off when the probe is added to the master mix and should remain off until the samples are placed in the Smart Cycler instrument. • The quantity of reagents needed for master mix is described below for 4 and 5 person group. Be sure to prepare master mix using the protocol designed for your group size. As a rule, make one extra reactions worth of master mix for every 10 reactions to ensure you will have enough. For example: for 5 reactions, prepare master mix for 6, for 15 reactions prepare master mix .2for 17, for 25 reactions prepare master mix for 28, and so on.

Table3. Real-time RT-PCR reaction mix volumes and conditions for type A influenza (MA gene).

Component

Master mix for 4 person

group

Master mix for 5 person

group

Volume Per Reaction

Final Concentration

H2O 83.4 μl 97.3 6.95μl 5X 60.0 70.0 5 1X

25mM MgCl2 15.0 17.5 1.25 3.75 mM Enzyme Mix 12.0 14.0 1

Forward Primer 6.0 7.0 0.5 10 pmol Reverse Primer 6.0 7.0 0.5 10 pmol

dNTP’s 9.6 11.2 0.8 320 μM ea. dNTP

Probe 6.0 7.0 0.5 0.15 μM Rnase Inhibitor 6.0 7.0 0.5 13 units

MM per rxn 17 17 Template 8 8

Total 25 25 2. Mix by vortexing for 3-5 seconds and centrifuge briefly. 3. Add 17μl of the master mix to each of your Smart Cycler tubes (add the mix to the bottom of the cup at the top of the reaction tube). 4. Add 8μl of template to the Smart Cycler tubes, close and label each tube.

Step 4 notes: • The template for the positive controls is in vitro transcribed RNA from the target gene • The template for the negative controls is nuclease free water. 5. Centrifuge the reaction tubes briefly in the Smart Cycler centrifuge. 6. Place the reaction tubes into the Smart Cycler and run with assay specific program. III. ANALYSIS OF RESULTS On the Smart Cycler the default minimum increase in fluorescence for a sample to be classified as positive by the software is 30 units. Because this is an arbitrary threshold, any samples which have an increase in fluorescence between 20 and 40 should be considered suspect and should be retested. Any questionable samples should be re-tested. If results of the second test are unsatisfactory additional sampling from the flock or premises should be considered if possible. Determining the results • Check the controls • Check each sample • Record the cycle threshold (Ct) values

– If a sample has no cycle threshold values (0.00) it is negative • Determine if there are any suspect samples

– Weak positives- Ct values >35 Suspect samples • For AIV or NDV a farm or premise is never considered positive based on one positive RTPCR result

– Epidemiology- dangerous contact – Clinical condition – Other positive diagnostic test

• Directigen (AIV) • Virus isolation • A second RT-PCR test for a different target

– AIV subtype specific – NDV- vNDV or vaccine virus specific

• Are other samples from the same farm positive? • Are there enough samples from the farm? • Were the controls valid?

Real-time PCR Basics The general principle of real-time PCR is the same as standard PCR; however the reaction product can be monitored in real-time with a fluorogenic probe. There are several types of detection systems for real-time PCR: hydrolysis probes, hybridization probes, molecular beacons and double stranded DNA binding dyes, among others. This assay utilizes hydrolysis probes. In the hydrolysis probe system, a DNA probe which binds the PCR product is added to the PCR reaction. The DNA probe has a fluorogenic reporter dye on one end and a quencher dye on the other end (figure 1). As the target PCR product increases the probe binds the amplicons and reporter dye is cleaved from the 5’ end of the probe by taq polymerase (due to 5’ exonuclease activity). As the reporter is cleaved from more and more probe molecules the fluorescence signal from the reaction increases. The fluorescence signal is monitored every cycle, revealing increases in the PCR product as it occurs. Additional information about Real-time PCR, primers and probes can be found at www.operon.com and www.idtdna.com.

Figure 1. Hydrolysis probe mechanism. a. The probe ( ) binds the PCR product ( ) during amplification. b. The polymerase ( ) runs into the probe during synthesis of the PCR product. c. Taq polymerase cleaves the reporter dye from the probe, increasing the detectable

JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/00/$04.0010

Nov. 2000, p. 4096–4101 Vol. 38, No. 11

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Detection of Influenza A Viruses from Different Species by PCRAmplification of Conserved Sequences in the Matrix Gene

RON A. M. FOUCHIER,* THEO M. BESTEBROER, SANDER HERFST, LIANE VAN DER KEMP,GUUS F. RIMMELZWAAN, AND ALBERT D. M. E. OSTERHAUS

National Influenza Center and Department of Virology, Erasmus University, Rotterdam, The Netherlands

Received 11 May 2000/Returned for modification 27 July 2000/Accepted 5 September 2000

The recently raised awareness of the threat of a new influenza pandemic has stimulated interest in thedetection of influenza A viruses in human as well as animal secretions. Virus isolation alone is unsatisfactoryfor this purpose because of its inherent limited sensitivity and the lack of host cells that are universallypermissive to all influenza A viruses. Previously described PCR methods are more sensitive but are targetedpredominantly at virus strains currently circulating in humans, since the sequences of the primer sets displayconsiderable numbers of mismatches to the sequences of animal influenza A viruses. Therefore, a new set ofprimers, based on highly conserved regions of the matrix gene, was designed for single-tube reverse transcrip-tion-PCR for the detection of influenza A viruses from multiple species. This PCR proved to be fully reactivewith a panel of 25 genetically diverse virus isolates that were obtained from birds, humans, pigs, horses, andseals and that included all known subtypes of influenza A virus. It was not reactive with the 11 other RNAviruses tested. Comparative tests with throat swab samples from humans and fecal and cloacal swab samplesfrom birds confirmed that the new PCR is faster and up to 100-fold more sensitive than classical virus isolationprocedures.

Migratory birds and waterfowl are thought to serve as thereservoir for influenza A viruses in nature (24). To date, influ-enza A viruses representing 15 hemagglutinin (HA) and nineneuraminidase (NA) subtypes have been detected in wild birdsand poultry throughout the world (19, 24). Since the generalhuman population is serologically naive with respect to mostavian HA and NA antigens, influenza A viruses of avian originpose a threat that is at the basis of new pandemics in humans(4, 24). For some time it was thought that avian influenzaviruses could be transmitted to humans only through coinfec-tion and genetic reassortment of avian and swine or humaninfluenza viruses in pigs (4, 13, 22, 24, 25). However, the recentzoonotic events in Hong Kong and mainland China caused byH5N1 and H9N2 influenza viruses suggest that avian influenzaviruses can be transmitted directly to humans as well (5, 8–10,15). The link between human influenza and the avian influenzavirus reservoir has boosted the public health-related and sci-entific interest in the prevalence, variability, and zoonotic po-tential of avian influenza viruses.

Although the routine procedures for the detection of humaninfluenza A viruses described to date, including in vitro virusisolation, immunofluorescence (IF), and PCR-based assays,are powerful tools, they may be less effective for the detectionof influenza viruses of avian and porcine origin. The pheno-typic and genetic heterogeneities of the latter viruses mayresult in a false-negative diagnosis of influenza A virus infec-tion by in vitro cell culture or current protocols for PCR anal-ysis. Importantly, sporadic zoonotic events of influenza A virusinfection may remain undetected as a result of such false-negative diagnoses.

The aim of this study was to set up a rapid and sensitive PCRmethod for the screening of clinical specimens for the presence

of phenotypically and genotypically diverse influenza A viruses.To this end, we have designed a primer set for PCR-baseddetection of influenza A viruses that was validated with clinicalspecimens and a panel of influenza A virus strains representingall known HA and NA subtypes obtained from a variety of hostspecies and from different geographical locations. The efficacyof this PCR-based screening of samples from avian and humanorigin was compared with classical isolation of influenza Avirus in embryonated chicken eggs or mammalian cell culture.We conclude that this PCR, based on the detection of genesegment 7 of influenza A virus, is fast, sensitive, and specificand is suitable for all genetic variants of influenza A virusknown to date.

MATERIALS AND METHODS

Design of oligonucleotides. PCR primers were designed on the basis of se-quence information obtained from the Influenza Sequence Database at LosAlamos National Laboratories, Los Alamos, N.M. (http://www.flu.lanl.gov). Toidentify conserved sequences in the influenza virus gene segments, entropy plotswere created with the Bioedit software package (available through http://www.mbio.ncsu.edu/RNaseP/info/programs/BIOEDIT/bioedit.html). Becausethe HA and NA genes are genetically diverse and sequence information on thePA, PB1, and PB2 polymerase genes is limited (less than 100 sequence entriesare available from the database, including partial sequences) only (partial) se-quences representing gene segments 5, 7, and 8 encoding nucleoprotein, matrix,and nonstructural proteins, respectively, were analyzed. The degree of hetero-geneity was expressed as entropy as defined by Shannon: H (1) 5 2Sf(b, 1) ln[f(b, 1)], where H (1) is the uncertainty at position 1, b represents a residue outof the allowed choices for the sequence in question (A, C, G, T, 2), and f(b, 1)is the frequency at which residue b is found at position 1 (16, 21). Oligonu-cleotides M52C (59-CTT CTA ACC GAG GTC GAA ACG-39) and M253R(59-AGG GCA TTT TGG ACA AAG/T CGT CTA-39) were designed for PCRamplification of influenza A virus matrix gene sequences, and the biotinylatedoligonucleotide Bio-M93C (59-CCG TCA GGC CCC CTC AAA GCC GA-39)was synthesized for hybridization purposes (Eurogentec, Seraing, Belgium).

Specimens. Cloacal swab specimens were collected from ducks (widgeon[Mareca penelope], gadwall [Mareca strepera], and mallard [Anas plathyrhynchos])at a marshaling lake in Lekkerkerk, The Netherlands, and droppings as well ascloacal swab specimens were collected from geese (greylag goose [Anser anser],white-fronted goose [Anser albifrons albifrons], barnacle goose [Branta leucopsis],and brent goose [Branta bernicla]) in Groningen and Eemdijk, The Netherlands,between 1997 and 1999. Cloacal swab specimens and droppings were collectedfrom shorebirds at Oland, Sweden, in the spring of 1999. Cotton swabs were used

* Corresponding author. Mailing address: Department of Virology,Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam,The Netherlands. Phone: 31 10 4088066. Fax: 31 10 4089485. E-mail:fouchier@viro.fgg.eur.nl.

4096

for sampling and were subsequently stored in transport medium (23). Throatswab specimens collected from humans were also stored in transport medium.The samples were stored at 4°C for a few days, at 220°C for less than a week, orat 270°C for extended periods of time. Transport medium consisted of Hanksbalanced salt solution supplemented with 10% glycerol, 200 U of penicillin perml, 200 mg of streptomycin per ml, 100 U of polymyxin B sulfate per ml, 250 mgof gentamicin per ml, and 50 U of nystatin per ml (all from ICN, Zoetermeer,The Netherlands).

RNA isolation. RNA was isolated with a high pure RNA isolation kit (RocheMolecular Biochemicals) according to the instructions from the manufacturer,with minor modifications. A 0.2-ml sample was homogenized by vortexing andwas subsequently lysed with 0.4 ml of lysis-binding buffer to which poly(A)(Roche Molecular Biochemicals) was added as a carrier to 1 mg/ml. After bindingto the column, DNase I digestion, and washing, the RNA was eluted in 50 ml ofnuclease-free double-distilled water preheated to 80°C.

PCR. The reverse transcription (RT) and PCRs were optimized with respect toenzymes, primer sets, and concentrations of reagents as well as cycling param-eters. Samples were amplified in a one-step RT-PCR in a final volume of 25 mlcontaining 50 mM Tris z HCl (pH 8.5), 50 mM NaCl, 7 mM MgCl2, 2 mMdithiothreitol, 1 mM each deoxynucleoside triphosphate at a concentration of 1mM, each oligonucleotide at a concentration of 0.4 mM, 2.5 U of recombinantRNAsin, 10 U of avian myeloblastosis virus reverse transcriptase, 2.5 U ofAmpli-Taq DNA polymerase (all enzymes were from Promega Benelux B.V.,Leiden, The Netherlands), and 5 ml of RNA. Thermocycling was performed in anMJ PTC-200 apparatus with the following cycling conditions: 30 min at 42°C and4 min at 95°C once and then 1 min at 95°C, 1 min at 45°C, 3 min at 72°C 40 times.Each reaction was analyzed by agarose gel electrophoresis and ethidium bromidestaining (10 ml/sample), followed by Southern blot hybridization (2) or dot blothybridization (5 ml/sample).

Dot blot hybridization. Five microliters of each of the PCR products wasincubated for 5 min at room temperature with 45 ml of 10 mM Tris z HCl (pH8.0), 1 mM EDTA, and 50 ml of 1 M NaOH for denaturation. The samples weretransferred to prewetted Hybond N1 membranes (Amersham Pharmacia Bio-tech Benelux, Roosendaal, The Netherlands) with a dot blot apparatus whileapplying vacuum. The samples were then treated for 3 min with 0.1 ml of 1 MTris z HCl (pH 8.0), after which vacuum was again applied for 10 s and themembrane was removed from the apparatus. The blots were washed three timesfor 10 min each time with 0.3 M NaCl–30 mM sodium citrate (pH 7), dried, andstored at 4°C. The blots were prehybridized for 5 min at 55°C in 23 SSPE (0.3M NaCl, 20 mM NaH2PO4, 2 mM EDTA [pH 7.4]) and 0.1% sodium dodecylsulfate (SDS), after which biotinylated oligonucleotide probe Bio-M93C wasadded to 2 pmol/ml and hybridization was continued for 45 min at 55°C. Theblots were washed twice for 10 min each time at 55°C with hybridization bufferand transferred to 23 SSPE with 0.5% SDS, after which streptavidin-peroxidase(Roche Molecular Biochemicals) was added to 0.125 U/ml and the mixture wasincubated for 45 min at 42°C. The blots were washed for 10 min at 42°C in 23SSPE–0.5% SDS, 10 min at 42°C in 23 SSPE–0.1% SDS, and 10 min at roomtemperature in 23 SSPE, after which the samples were visualized with enhancedchemiluminescence detection reagents and by exposure to hyperfilm (AmershamPharmacia Biotech Benelux) for 5 to 60 s.

Virus isolation and propagation. The influenza A viruses listed in Table 1 havebeen described earlier and were kindly provided by R. G. Webster (14, 19). Allof these viruses had been isolated and propagated in the allantoic cavities of11-day-old embryonated chicken eggs (12). Influenza virus A/Netherlands/18/94has been described previously (18). Influenza A virus strains not listed in Table1 were isolated and propagated in Madin-Darby canine kidney (MDCK) cells ortertiary monkey kidney (tMK) cells derived from cynomolgus macaques (Macacafascicularis) (7, 17). Virus stocks were titrated by end point dilution in MDCK ortMK cells, and the 50% tissue culture infective doses (TCID50s) were calculatedas described previously (17). The HA titers in the virus stocks were determinedwith turkey erythrocytes by standard procedures (17). Virus isolates were char-acterized by hemagglutination inhibition assays with subtype-specific hyperim-mune rabbit antisera raised against HA and NA preparations of the virus isolateslisted in Table 1 (20).

Human respiratory syncytial virus (HRSV) was grown in HEp-2 cells, mumpsand measles viruses were grown in Vero cells, human parainfluenza virus (PIV)types 1 through 4 (PIV-1 through PIV-4) and influenza B virus were grown intMK cells, and Sendai virus, simian parainfluenza virus type 5 (SV5), and New-castle disease virus (NDV) were grown in embryonated chicken eggs. The virustiters of these stocks typically ranged from 104 to 106 TCID50s/ml.

RESULTS

Design of oligonucleotides for PCR detection of influenza Aviruses. Avian and mammalian influenza A virus nucleotidesequences available from the influenza sequence database(http://www.flu.lanl.gov) were compared to the sequences ofpreviously described primer sets Mx1 and Mx2 (3), Fam1 andFam2 (1), and NS486C and NS637R (6, 7) to analyze theirpotential for the detection of genetically diverse influenza A

viruses. The variability between the influenza A virus nucleo-tide sequences and each position in the potential PCR primerswas calculated by using the entropy algorithm available fromthe Bioedit software package (16, 21). Although each of theprimer sequences was based on a relatively conserved domainof gene segments 7 and 8 of influenza A virus, considerableheterogeneity was observed for each of the oligonucleotide sets(Fig. 1). The 39 ends of oligonucleotides are of the greatestimportance for the successful amplification by PCR. Of allthree published primer sets (Fig. 1A to F), at least one of theoligonucleotides displayed considerable numbers of mis-matches with the sequences in the database. Since such mis-matches may lead to false-negative PCR results, we designednew primer sets based on segment 7 of influenza A virus, whichis relatively conserved compared to the other segments. Withinthe M1 coding sequence of gene segment 7, several regions(positions 32 to 93, 149 to 204, and 218 to 276) were identifiedthat are relatively conserved among influenza A virus strainsobtained from a variety of host species and from differentgeographical regions. Oligonucleotides M52C (nucleotide po-sitions 32 to 52), M93C (positions 71 to 93), and M253R(positions 253 to 276) (Fig. 1) were designed on the basis ofthese conserved regions of the influenza A virus genome. Al-though other conserved regions were identified in the NS2coding sequence of gene segment 8 and the M1 coding se-quence of segment 7, we found primers based on these se-quences to be less suitable for PCR amplification of selectedinfluenza A virus strains (data not shown).

Sensitivity and specificity of influenza A virus PCR. RNAwas isolated from 0.2 ml of allantoic fluid containing the in-fluenza A viruses shown in Table 1, and the equivalent of 4 mlof allantoic fluid was used for amplification by PCR withprimer set M52C-M253R. For each of the virus strains tested,a band of 244 bp was amplified and was easily visualized on a1% agarose gel stained with ethidium bromide (Fig. 2). Hy-

TABLE 1. Virus isolates used for the validation of PCR-baseddetection of influenza A virus

Influenza A virus strain HAsubtype

NAsubtype

HAtiter

Lane no.(Fig. 2)

A/Puerto Rico/8/34 1 1 384 1A/Fort Monmouth/1/47 1 1 384 2A/Swine/Shope/56 1 1 512 3A/Duck/Alberta/35/76 1 1 768 4A/Singapore/1/57 2 2 256 5A/Hong Kong/1/68 3 2 512 6A/Equine/Miami/1/63 4 8 256 7A/Duck/Ukraine/1/63 5 8 512 8A/Duck/Czechoslovakia/1/56 6 6 256 9A/Tern/South Africa/61 5 3 256 10A/Duck/Hong Kong/205/77 5 3 128 11A/Turkey/Massachusetts/65 6 —a 512 12A/Shearwater/Australia/1/72 6 5 192 13A/Equine/Prague/1/56 7 7 1024 14A/Seal/Massachusetts/1/80 7 7 128 15A/Turkey/Ontario/6118/68 8 4 128 16A/Turkey/Wisconsin/1/66 9 2 384 17A/Chicken/Germany/49 10 7 384 18A/Duck/England/1/56 11 6 256 19A/Duck/Memphis/546/76 11 9 768 20A/Duck/Alberta/60/76 12 5 128 21A/Gull/Maryland/704/77 13 6 256 22A/Mallard/Gurjev/263/82 14 — 768 23A/Duck/Australia/341/83 15 8 256 24A/Shearwater/West Australia/2576/79 15 9 512 25

a —, NA subtype unknown.

VOL. 38, 2000 PCR-BASED DETECTION OF INFLUENZA A VIRUSES 4097

bridization of dot blots with the internal biotinylated oligonu-cleotide probe M93C also resulted in clear signals for each ofthe influenza A virus strains tested.

We next compared the sensitivity of this PCR with viruspropagation in cell cultures. A stock of influenza virus A/Neth-erlands/18/94 (H3N2) was generated in tMK cells. This virusstock contained 107 TCID50s of influenza A virus per ml ofculture supernatant, as determined with tMK and MDCK cells(17). Serial 10-fold dilutions of virus were made in transportmedium, and RNA was isolated for use in PCR analysis, aga-rose gel electrophoresis, or dot blot hybridization. The ex-pected DNA fragment of 244 bp was visible on an agarose gelstained with ethidium bromide when the RNA equivalent of0.2 TCID50 of influenza A virus was used as input in the PCR(Fig. 3, lane 8). By using dot blots and hybridization, 0.02TCID50 of influenza A virus was found to be the detection limitof the assay (Fig. 3, lane 9, and data not shown). Similar resultswere obtained with a second influenza A virus isolate, and such

results were found to be reproducible (data not shown). Thesedata indicate that our PCR procedure is up to 100-fold moresensitive than virus propagation in MDCK and tMK cells.

To test the specificities of our PCR primers, RNA was iso-lated from stocks of a number of RNA viruses, followed byPCR amplification and gel electrophoresis or dot blot hybrid-ization. RNA was isolated from 0.2 ml of virus stocks contain-ing either influenza B virus, HRSV, PIV-1 through PIV-4,simian parainfluenza virus type 5 (SV5), NDV, mumps virus,measles virus, or Sendai virus. One-tenth of the RNA, repre-senting the equivalent of 20 ml of virus stock ranging in titerfrom 104 to 106 TCID50s/ml, was used for PCR. Upon agarosegel electrophoresis, weak bands and smears of bands rangingfrom 150 to 400 bp in length were observed after PCR ampli-fication of some of the virus samples (PIV-1, -2, and -3, NDV,mumps virus, and influenza B virus), presumably as a result ofnonspecific amplification of the high levels of viral RNApresent in these samples. However, upon hybridization of dotblots with the biotinylated oligonucleotide M93C, all RNA

FIG. 1. Entropy plots of oligonucleotide-annealing sites in human and animal influenza A virus sequences available from the influenza virus sequence database. Thesequences recognized by oligonucleotides Mx1, Fam1, NS486C, Mx2, Fam2, NS637R, M52C, M253R, and M93C were compared to all available influenza A virussequences (n 5 189, 189, 234, 203, 204, 249, 175, 215, and 189, respectively), and their heterogeneities are displayed in panels A through I, respectively. Oligonucleotidepositions are given in the 59 to 39 direction, with position 1 being the extreme 59 nucleotide. Asterisks indicate primer positions with degeneracy in the designedoligonucleotides. Oligonucleotides M52C, M253R, and M93C were designed in the present study.

FIG. 2. PCR analysis of the influenza A viruses, listed in Table 1, whichoriginated from different hosts and geographical locations. RNA was isolatedfrom influenza A viruses grown in embryonated chicken eggs, followed by PCRanalysis and agarose gel electrophoresis (top panels) or dot blot analysis (bottompanels). Lanes 1 to 25, see Table 1; lane 26, negative control.

FIG. 3. Sensitivity of detection of influenza A virus RNA by PCR. RNA wasisolated from 0.2 ml of 10-fold serial dilutions of influenza virus A/Netherlands/18/94 (107 TCID50s/ml) and was used for PCR analysis followed by agarose gelelectrophoresis and ethidium bromide staining (top panel) or dot blot analysis(bottom panel). Lane 1, negative control; lanes 2 to 9, dilution series represent-ing the equivalent of 2 3 105 to 0.02 TCID50s per sample. Samples containingless than 0.02 TCID50 were negative by PCR and dot blot analysis (data notshown).

4098 FOUCHIER ET AL. J. CLIN. MICROBIOL.

virus samples except for that with influenza A virus were neg-ative (Fig. 4).

Detection of influenza A virus in human throat swab sam-ples. Throat swab samples sent to the virus diagnostic labora-tory at Erasmus University Medical Center are routinely testedfor the presence of influenza A virus by direct IF (DIF) andinoculation in MDCK or tMK cell cultures in combination withIF (7). For a selection of influenza A virus-positive throat swabsamples obtained in the 1994-1995 influenza season, influenzaA virus titers were determined by end point dilution and in-oculation of tMK cells. A selection of influenza A virus-posi-tive (n 5 13) and influenza A virus-negative (n 5 26) sampleswas coded and tested blindly by PCR and dot blot hybridiza-tion. All influenza A virus-positive samples, with titers rangingfrom 0 to 105.75 TCID50s per ml of throat swab sample, werepositive upon agarose gel electrophoresis and dot blot hybrid-ization (Fig. 5). One of the influenza A virus PCR-positivesamples (lane 6) tested negative upon inoculation of mamma-lian cell cultures (hence, 0 TCID50). This sample had beenfound to be influenza A virus positive by DIF with the cellspresent in the throat swab sample (7), but no virus could beisolated. Of 26 negative control samples (13 were influenza B

virus positive and 13 were influenza A and B virus negative inmammalian cell cultures), 24 were negative upon PCR and dotblot analyses. Two of the swabs were negative for influenza Avirus in mammalian cell culture and by IF but yielded veryweak signals after PCR and dot blot hybridization (lanes 9 and30). These weak dot blot signals may be due to backgroundhybridization or the presence of very small amounts of influ-enza A virus RNA in the throat swabs.

Detection of influenza A virus in bird samples. We nexttested the suitability of the PCR for avian influenza A virusscreening of cloacal swab and dropping samples from ducks,geese, and shorebirds collected in The Netherlands and Swe-den. Because PCR screening appeared to be up to 100-foldmore sensitive than virus isolation (see above) and to reducecost and workload, the numbers of RNA isolations and PCRanalyses were reduced by making pools of five samples each(40 ml per sample). Between each five pooled samples, a neg-ative control consisting of transport medium was inserted tocheck for contamination during processing of the samples.Among the 235 pools of samples representing 1,175 individualspecimens, RNA isolation, PCR, and Southern or dot blothybridization revealed the presence of influenza A virus in 19of them (the results of the analysis of 38 of these pools is shownin Fig. 6). RNA was then isolated from each of the individualsamples present in these 19 pools, revealing that all except 1pool contained a single positive bird sample; the one exceptioncontained two positive samples.

Each of the 20 positive individual samples was used to in-

FIG. 4. Specificity of detection of influenza A virus RNA by PCR. RNA wasisolated from virus stocks and was used for PCR analysis and subsequent agarosegel electrophoresis (top panel) or dot blot hybridization (bottom panel). Lanes:1, HRSV; 2, PIV-1; 3, PIV-2; 4, PIV-3; 5, PIV-4; 6, Sendai virus; 7, SV5; 8, NDV;9, mumps virus; 10, measles virus; 11, influenza B virus; 12, influenza A virus.

FIG. 5. PCR-based detection of influenza A virus in 39 human throat swabsamples. Throat swab samples that were tested previously for the presence ofinfluenza A virus by classical screening methods (7) were randomized and testedblindly by PCR. RNA was isolated from 0.2 ml of a throat swab sample and wasused for PCR and dot blot analysis. Lanes 1, 4, 7, 8, 13, 16, 18, 23, 24, 30, 34, 35,and 38, influenza virus-negative samples; lanes 2, 5, 9, 10, 12, 14, 15, 20, 21, 22,25, 29, and 31, influenza B virus-positive samples; lane 40, 10 TCID50s ofinfluenza virus A/Netherlands/18/94 as a positive control; lanes 3, 6, 11, 17, 19,26, 27, 28, 32, 33, 36, 37, and 39, influenza A virus-positive samples in which virustiters determined in MDCK cells were 105.75, 0, 103.5, 102.25, 100.75, 104.25, 100.75,103.75, 104.25, 105.25, 104.5, 105.75, and 103.5 TCID50s/ml respectively.

FIG. 6. PCR-based detection of influenza A virus in a representative set ofavian cloacal swab and dropping samples. RNA was isolated from 0.2 ml of 38pooled samples, each consisting of five individual bird samples, and was used forPCR and Southern blot analysis. Lanes 1, 11, 21, 31, and 41, positive controlsrepresenting 10 TCID50s of influenza virus A/Netherlands/18/94; lanes 7, 14, 20,27, 34, 40, and 47, negative controls; lanes 2 to 5, duck cloacal swab samples;lanes 6, 8 to 10, 12, 13, 15 to 19, 22 to 26, and 28 to 30, goose dropping samples;lanes 32, 33, 35 to 39, 42 to 46, and 48 to 50, goose cloacal swab samples. Eachof the pools represented in lanes 13, 15, 23, 30, 36, 39, 43, and 44 was found tocontain a single positive individual bird sample. Virus was isolated in embryo-nated chicken eggs from samples represented in lanes 13, 15, 23, 30, 39, and 43but not from those represented in lanes 35, 36, and 44.

VOL. 38, 2000 PCR-BASED DETECTION OF INFLUENZA A VIRUSES 4099

oculate two to four embryonated chicken eggs from which theallantoic fluids were collected, pooled, and inoculated a secondtime in duplicate in embryonated chicken eggs (blind passage).For 15 of 20 PCR-positive samples we were able to isolateinfluenza A virus in eggs. For the other five samples, whichappeared to contain less virus, as judged by the intensity of thesignals on dot blots (e.g., lanes 35, 36, and 44 in Fig. 6), noinfluenza A virus could be isolated even upon blind passage inembryonated chicken eggs.

To test the possibility that the PCR analysis would givefalse-negative results compared to virus isolation in eggs, 243individual PCR-negative cloacal swab and dropping sampleswere inoculated into two to four embryonated chicken eggseach, followed by a blind passage of the pooled allantoic fluidsin duplicate. We were unable to isolate influenza A virus fromthese PCR-negative samples, indicating that no false-negativeresults were obtained by PCR analysis. Inoculation of tMK andMDCK cell cultures with 212 random PCR-negative individualbird samples also did not reveal additional influenza A virus-positive samples. In fact, these cell lines were found to be lesssusceptible to avian influenza A virus than embryonatedchicken eggs were (data not shown).

DISCUSSION

PCR-based methods for virus detection have been describedfor many clinically relevant viruses. The sensitivities and spec-ificities of PCR-based methods are most critically determinedby the choice of primer sequences. The sequences of theprimer sets described earlier for PCR-based detection of in-fluenza A virus may be appropriate for the detection of virusstrains currently circulating in humans (1, 3, 6, 7) but displayconsiderable numbers of mismatches when they are comparedwith the sequences of animal influenza A viruses. We haveused an extensive amount of the sequence information avail-able for influenza A virus to design a new PCR primer set fordiagnostic purposes. Primers M52C and M253R and probeM93C span conserved sequences in gene segment 7 of influ-enza A virus and have no homology to nucleotide sequencesfrom other species available from GenBank (http://www.ncbi.nlm.nih.gov). Our experimental data confirmed that PCRamplification and dot blot analyses with this set of primers doesnot pick up cross-reacting host-derived sequences or otherRNA viruses and is suitable for detection of a wide variety ofinfluenza A virus strains. The limited variability in influenza Avirus sequences spanning the primer sequences is mostly con-fined to the 59 ends of the oligonucleotides and therefore isunlikely to obscure PCR amplification. Indeed, we successfullyamplified the genomes of virus isolates with mismatches inthese primer sequences that were included in the viruses shownin Table 1 and Fig. 2.

On the basis of the results of titration experiments as well ason analyses of clinical specimens, we conclude that the PCR-based method is more sensitive (up to 100-fold) than virusisolation in eggs or mammalian cell cultures. This is not sur-prising in view of the sensitivity of PCR-based assays in generaland the low ratio of infectious units to physical particles forRNA viruses such as influenza A virus. Perhaps as a result ofthe high sensitivity, we detected influenza A virus in a humanthroat swab sample from which no virus could be isolated.Individual cells isolated from this throat swab sample werepositive upon DIF analysis, confirming influenza A virus infec-tion.

An additional advantage of the PCR-based method is itsvalue in the identification of influenza A viruses from differentspecies. Because of differences in cellular tropism between

avian, human, and swine influenza A viruses, a single cell typefor virus isolation for diagnostic purposes is not available.Continuous and primary cell lines obtained from a variety ofanimal species and embryonated chicken eggs are routinelyused for isolation of influenza A viruses. Using the PCR-basedmethod, we have detected many influenza A viruses in birdsamples that could not be isolated in mammalian cell culturesand some that could not be isolated in embryonated chickeneggs. Presumably, this failure was due to a combination of lowvirus titers in the original specimens and the limited suscepti-bilities of the target cells to certain influenza A virus strains. Asa national influenza center, we occasionally receive specimensfrom humans from which no virus can be isolated in mamma-lian cell cultures but that are readily found to be influenza Avirus positive by this PCR approach (data not shown).

One disadvantage of PCR-based assays is that it is difficult toassess if weak positive PCR results (e.g., Fig. 5, lanes 9 and 30,and Fig. 6, lanes 35, 36, and 44) are the result of backgroundhybridization or low virus titers in the original samples becauseof the lack of confirmation assays that are as sensitive as PCR-based methods. Therefore, it is of great importance that suf-ficient negative controls be included to determine a cutoffvalue for background hybridization. In addition, we routinelyuse 10-fold serial dilutions of a titrated influenza A virus stockas input material in our PCR-based assays to provide a semi-quantitative estimate of variability between independent as-says. Both sets of controls will aid in the determination of acutoff value for background hybridization and weak positivesamples.

By PCR-based assays, diagnosis of influenza A virus infec-tion can be achieved within a single working day, which issignificantly faster than the time to diagnosis of infection byclassical methods. By virus culture approaches, positive resultsmay be obtained in 24 h or more after inoculation, but adefinite negative diagnosis may require culture for up to 2weeks. The availability of NA inhibitors for the treatment ofinfluenza virus infection may demand more rapid diagnosis ofvirus infection in the future. The benefit of these new drugsappears to depend heavily on the early start of treatment, i.e.,within 2 days after the onset of disease (11).

Taken together, our data indicate that the newly designedPCR offers a more sensitive and faster tool for the diagnosis ofhuman influenza A virus infection than virus isolation. Becauseof the better matching primers, it can be expected that for thedetection of animal influenza A viruses this PCR is also moresuitable than previous PCR protocols (1, 3, 7).

ACKNOWLEDGMENTS

We thank John de Boer, Hans Zantinge, Dick Jonkers, Bjorn Olsen,and their colleagues for collection of bird samples, Rob Webster forproviding influenza A virus isolates, Jan Groen and Bernadette vanden Hoogen for samples from RNA viruses, and Jan de Jong forcritically reading the manuscript. R.A.M.F. is a fellow of the RoyalDutch Academy of Arts and Sciences.

This work was made possible in part through a grant from the DutchMinistry of Agriculture and from the Foundation for Respiratory Vi-rus Infections (SRVI).

REFERENCES

1. Atmar, R. L., B. D. Baxter, E. A. Dominguez, and L. H. Taber. 1996. Com-parison of reverse transcription-PCR with tissue culture and other rapiddiagnostic assays for detection of type A influenza virus. J. Clin. Microbiol.34:2604–2606.

2. Brown, T. 2000. Analysis of DNA sequences by blotting and hybridization, p.2.9.1–2.9.15. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G.Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecularbiology, suppl. 45. John Wiley & Sons, Inc., New York, N.Y.

3. Cherian, T., L. Bobo, M. C. Steinhoff, R. A. Karron, and R. H. Yolken. 1994.

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Use of PCR-enzyme immunoassay for identification of influenza A virusmatrix RNA in clinical samples negative for cultivable virus. J. Clin. Micro-biol. 32:623–628.

4. Claas, E. C., and A. D. Osterhaus. 1998. New clues to the emergence of flupandemics. Nat. Med. 4:1122–1123.

5. Claas, E. C., A. D. Osterhaus, R. van Beek, J. C. De Jong, G. F. Rimmel-zwaan, D. A. Senne, S. Krauss, K. F. Shortridge, and R. G. Webster. 1998.Human influenza A H5N1 virus related to a highly pathogenic avian influ-enza virus. Lancet 351:472–477.

6. Claas, E. C., M. J. Sprenger, G. E. Kleter, R. van Beek, W. G. Quint, and N.Masurel. 1992. Type-specific identification of influenza viruses A, B and C bythe polymerase chain reaction. J. Virol. Methods 39:1–13.

7. Claas, E. C., A. J. van Milaan, M. J. Sprenger, M. Ruiten-Stuiver, G. I.Arron, P. H. Rothbarth, and N. Masurel. 1993. Prospective application ofreverse transcriptase polymerase chain reaction for diagnosing influenzainfections in respiratory samples from a children’s hospital. J. Clin. Micro-biol. 31:2218–2221.

8. de Jong, J. C., E. C. Claas, A. D. Osterhaus, R. G. Webster, and W. L. Lim.1997. A pandemic warning? Nature 389:554.

9. Guan, Y., K. F. Shortridge, S. Krauss, and R. G. Webster. 1999. Molecularcharacterization of H9N2 influenza viruses: were they the donors of the“internal” genes of H5N1 viruses in Hong Kong? Proc. Natl. Acad. Sci. USA96:9363–9367.

10. Guo, Y. J., S. Krauss, D. A. Senne, I. P. Mo, K. S. Lo, X. P. Xiong, M.Norwood, K. F. Shortridge, R. G. Webster, and Y. Guan. 2000. Characteri-sation of the pathogenicity of members of the newly established H9N2influenza virus lineage in Asia. Virology 267:279–288.

11. Hayden, F. G., A. D. Osterhaus, J. J. Treanor, D. M. Fleming, F. Y. Aoki,K. G. Nicholson, A. M. Bohnen, H. M. Hirst, O. Keene, and K. Wightman.1997. Efficacy and safety of the neuraminidase inhibitor zanamivir in thetreatment of influenzavirus infections. GG167 Influenza Study Group.N. Engl. J. Med. 337:874–880.

12. Hinshaw, V. S., R. G. Webster, and B. Turner. 1978. Novel influenza Aviruses isolated from Canadian feral ducks: including strains antigenicallyrelated to swine influenza (Hsw1N1) viruses. J. Gen. Virol. 41:115–127.

13. Ito, T., J. N. Couceiro, S. Kelm, L. G. Baum, S. Krauss, M. R. Castrucci, I.Donatelli, H. Kida, J. C. Paulson, R. G. Webster, and Y. Kawaoka. 1998.Molecular basis for the generation in pigs of influenza A viruses with pan-demic potential. J. Virol. 72:7367–7373.

14. Matrosovich, M., N. Zhou, Y. Kawaoka, and R. Webster. 1999. The surfaceglycoproteins of H5 influenza viruses isolated from humans, chickens, andwild aquatic birds have distinguishable properties. J. Virol. 73:1146–1155.

15. Peiris, M., K. Y. Yuen, C. W. Leung, K. H. Chan, P. L. Ip, R. W. Lai, W. K.Orr, and K. F. Shortridge. 1999. Human infection with influenza H9N2.Lancet 354:916–917.

16. Pierce, J. R. 1980. An introduction to information theory: symbols, signalsand noise, 2nd ed. Dover Publications, Inc., New York, N.Y.

17. Rimmelzwaan, G. F., M. Baars, E. C. Claas, and A. D. Osterhaus. 1998.Comparison of RNA hybridization, hemagglutination assay, titration of in-fectious virus and immunofluorescence as methods for monitoring influenzavirus replication in vitro. J. Virol. Methods 74:57–66.

18. Rimmelzwaan, G. F., M. Baars, R. van Beek, G. van Amerongen, K. Lovgren-Bengtsson, E. C. Claas, and A. D. Osterhaus. 1997. Induction of protectiveimmunity against influenza virus in a macaque model: comparison of con-ventional and iscom vaccines. J. Gen. Virol. 78:757–765.

19. Rohm, C., N. Zhou, J. Suss, J. Mackenzie, and R. G. Webster. 1996. Char-acterization of a novel influenza hemagglutinin, H15: criteria for determi-nation of influenza A subtypes. Virology 217:508–516.

20. Schild, G. C., R. W. Newman, R. G. Webster, D. Major, and V. S. Hinshaw.1980. Antigenic analysis of influenza A virus surface antigens: considerationsfor the nomenclature of influenza virus. Brief review. Arch Virol. 63:171–184.

21. Schneider, T. D., and R. M. Stephens. 1990. Sequence logos: a new way todisplay consensus sequences. Nucleic Acids Res. 18:6097–6100.

22. Scholtissek, C., H. Burger, O. Kistner, and K. F. Shortridge. 1985. Thenucleoprotein as a possible major factor in determining host specificity ofinfluenza H3N2 viruses. Virology 147:287–294.

23. Sharp, G. B., Y. Kawaoka, S. M. Wright, B. Turner, V. Hinshaw, and R. G.Webster. 1993. Wild ducks are the reservoir for only a limited number ofinfluenza A subtypes. Epidemiol. Infect. 110:161–176.

24. Webster, R. G., W. J. Bean, O. T. Gorman, T. M. Chambers, and Y.Kawaoka. 1992. Evolution and ecology of influenza A viruses. Microbiol.Rev. 56:152–179.

25. Zhou, N. N., D. A. Senne, J. S. Landgraf, S. L. Swenson, G. Erickson, K.Rossow, L. Liu, K. J. Yoon, S. Krauss, and R. G. Webster. 1999. Geneticreassortment of avian, swine, and human influenza A viruses in Americanpigs. J. Virol. 73:8851–8856.

VOL. 38, 2000 PCR-BASED DETECTION OF INFLUENZA A VIRUSES 4101

Real-Time RT-PCR Detection of Avian Paramyxovirus-1 and Avian Influenza Virus

Jan PedersenAvian Section Diagnostic Virology Laboratory

National Veterinary Services LaboratoryAmes, Iowa 50010

Janice.c.pedersen@aphis.usda.gov

Real-Time RT-PCR

RNA Extraction

RRT-PCRSample: i.e. Swab material

Results analysis

Advantages of rRT-PCR for the Detection of AIV and ND

Speed –results in as little as 3 hrs.

Scalable - large numbers of samples can be processed

Sensitive in-vitro surveillance assay that can test many samples

Cost (~$8 sample)

Viable virus not necessary

Advantages of rRT-PCR for the Detection of AIV and ND

Reduced handling of potentially infectious material

Sensitivity similar to virus isolation

Specific

Can differentiate virulent NDV (vNDV) strains from vaccine strains or lentogenic APMV-1 strains

Can detect H5 and H7 AIV, but can not differentiate HPAIV from LPAIV

Reduced chance for cross-contamination vs. standard RT-PCR

Disadvantages of rRT-PCRFalse Positives

Very sensitive: Cross-contaminationNon-specific detectionAIV and APMV-1 assays have been validated to error on the side of false positive rather than false negatives results

Initial equipment investment is expensive

Will detect live or inactivated virusNot appropriate for environmental specimens

Disadvantages of rRT-PCR

False negativesInhibitory substances in sample

Internal controls to identify false negativeOverloading silica-gel columns with organic material

Template modification/degradationRNA fragile

Real-time RT-PCR

PCR product is detected in real-timeSequence specific probe

Taqman/ HydrolysisFRET/ HybridizationMolecular beaconsLux Primers

Non-sequence specific DNA binding dyesSYBR green

Less expensive assay systemNot pathogen specific

Isolation vs. DetectionIsolation of the etiological agent

Conducted in chicken embryos

Necessary for characterization and pathogenicity studies

Time needed - 3 to 14 days

Detection of Nucleic Acid

Isolation of RNA from swab or tissue specimens

Amplification and identification of RNA and not live virus

Virus may be infectious or non-infectious

Time needed – 3 hrs.

RRT-PCR for AIV

MatrixPrimers/probe

Will detect all 16 H subtypes (H1-16) of AIVDetects both HPAI and LPAIDetects Asian H5N1

H5 Primers/probeDetects most North American strains of H5 AIVDetects Asian H5N1Detects both HPAI & LPAI

H7 Primers/probeDetects most North Americans strains of H7 AIVDetects both HPAI & LPAI

Evaluation of H5 Subtype RRT-PCR Test for Asian H5N1

H5 test was originally designed primarily for North American isolatesCould identify Asian H5N1 viruses with lower sensitivitySequence analysis of Asian isolates showed good conservation with reverse primer and probe, but 4 mismatches with forward primerRedesigned H5 test to include forward primers optimized for both Asian and North American viruses

NA H5F TGACTATCCACAATACTCAEA H5F TGACTACCCGCAGTATTCA

APMV-1 RRT-PCR Assay

APMV-1 primer/probe

Target: Matrix gene

Will detect most APMV-1 isolatesVirulent NDVAvirulent vaccine strainsPPMV

Cree/CalMex-VFP-1primer/probeTarget: fusion gene cleavage site

Designed to detect the CA 2002/03 strain of vNDVWill detect most velogens and mesogens.Will not detect vaccine strainsWill detect some PPMV

Hydrolysis/Taqman probes

Taq

Taq

Reporter

Reporter

Quencher

Quencher

Primer 1

Primer 2

Hydrolysis or Taqman Probes

5’ to 3’ nuclease activity ofTaq DNA polymerase

R Q

R Q

R

Q

Primer 1

Primer 1Taq

Specimens

Swabs – Can be pooledTracheal or oralpharyngeal – 5/tube

Preferred specimen

Cloacal – 5/tubeLess sensitive than tracheal/oralpharyngeal

TissuesLung, spleen, kidney

Tissue pools – small pieces of tissue in viral transport media

Specimen Processing in LabAll Procedures should be carried out in a biological safety cabinet or other primary containment device

Specimen processing and RNA extractionBSC II

Reagent preparation and RNA transferBSC or PCR workstation

Wear gloves at all times during this procedurePowder-free

RNA Extraction

Isolates RNA from other materials in the sample

Removes inhibitory substances – may not eliminate all

Strong detergents inactivate RNases that will degrade RNA

Extraction

Obtaining high quality RNA is the 1st and most important step

Handling of specimen

Storage of isolated RNAStore in RNase – free solution24 hr. - Store at 4 C >24 hr. - Store at -70 C

RNA Extraction- Materials

2 test samples for each person1.5ml microfuge tubesExtraction pipets and tips- dedicated equipmentVacuum manifold and tubing

RNA Extraction- Materials

RNeasy Kit componentsRLT with BMERPE buffer with ethanol addedRW1 bufferNuclease free water2ml collection tubes2 RNeasy columnsElution tubes

RNA Extraction- RNeasy Kit

1. Add 500μl swab supernatant to a1.5 ml micro centrifuge tube

2. Add 500μl RLT buffer 3. Vortex for 15 sec.4. Pulse centrifuge 5. Add 500 μl 70% ethanol and vortex 15

sec.

RNA Extraction- RNeasy Kit6. Centrifuge lysed specimen @ 5,000 xg for 5 min.

7. Set-up the vacuum manifold: Place the appropriate number of RNeasy columns in the luer locks of the vacuum manifoldCover any empty positions with the luer caps supplied with the vacuum manifold.

8. Apply vacuum and add the entire sample/RLT/ethanol mixture to an RNeasy column for each sample.

RNA Extraction- RNeasy Kit

9. Wash by applying 700μl RW1 buffer to each column.

10. Wash again by applying 500μl RPE buffer to the column and repeat for a total of 2 washes with buffer RPE.

11. Shut off the vacuum and place each RNeasy column in a 2ml collection tube.

RNA Extraction- RNeasy Kit12. Centrifuge the column and collection tube for 2 minutes

at ~14 K xg and discard the collection tube.

13. Place the column in an elution tube (or 1.5ml microfuge tube)

14. Add 50μl nuclease free water to the column membrane and incubate at room temperature 1 minute.

15. Elute RNA by centrifuging for 1 minute at ~14K xg. Store at -70°C long term.

Real-time RT-PCR

RT- reverse transcription – 50 C°cDNA is produced from RNA template

PCR- polymerase chain reactioncDNA is amplifiedDNA is more stable and more easily amplified than RNA

Real-timePCR amplification is monitored in real-time and the amplicon is detected with a fluorogenic probe

Real-time RT-PCR Materials

For each lab groupSmart Cycler tube cooling blockSmart Cycler tubes RNA samplesDedicated pipets and aerosol resistant pipet tips 1.5ml tubes

Real-time RT-PCR Materials

A set of RT-PCR reagents Enzyme and RNase inhibitor in a bench top cooler5X bufferdNTPSPositive control (AIV M or H5 RNA)Nuclease free waterForward primer (AIV M+25 or H5+1456)Reverse primer (AIV M-124 or H5-1685)Probe (AIV M+64 or H5+1637)

Prepare the reaction master mix in a 1.5ml tube

6.0Rnase Inhibitor6.0Probe9.6dNTP’s6.0Reverse Primer6.0Forward Primer12Enzyme Mix

15.025mM MgCl2

605X83.4H2O

Volume in μlComponent

7.0Rnase Inhibitor7.0Probe

11.2dNTP’s7.0Reverse Primer7.0Forward Primer

14.0Enzyme Mix17.525mM MgCl2

705X97.3H2O

Volume in μlComponent

4 people/group 5 people/group

RNA TransferMix reagents by vortexing for 3-5 seconds and centrifuge

briefly.

Add 17μl of the master mix to each of your Smart Cycler tubes (add the mix to the bottom of the cup at the top of the reaction tube).

Add 8μl of template to the Smart Cycler tubes, close and label each tube as follows:1. Positive control: in vitro transcribed RNA from the target gene 2. Negative control: nuclease free water.3. test sample 14. test sample 2

Setting up rRT-PCR

Centrifuge the reaction tubes briefly in the Smart Cycler centrifuge.

Place the reaction tubes into the Smart Cycler and run with the “AIV Matrix” or “H5” program.

The program has already been programmed into the smart cycler

Results Interpretation

Log-linearbaseline

Primary Growth Curve

Baseline

Log-linear

Plateau

Log-linear

Curve entering Log-linear

baseline

baseline

Threshold set appropriately

Threshold set too low

Evaluation of Growth Curve

Positive

Negative

Results interpretation

Results interpretation

25

Results TableResults interpretation

Check the controls

Check background fluorescence

Check each sample individuallyThe computer is not always correct

Look for software artifacts

Software Artifacts Software Artifacts

Results interpretation

Record the cycle threshold (Ct) valuesIf a sample has no cycle threshold values (0.00) it is negative

Determine if there are any suspect samples

Weak positives- Ct values >35

Suspect samplesFor AIV or NDV a farm or premise is never considered positive based on one positive RT-PCR result

Epidemiology- dangerous contactClinical conditionOther positive diagnostic test

Directigen (AIV) Virus isolation A second RT-PCR test for a different target

AIV subtype specificNDV- vNDV or vaccine virus specific

Are other samples from the same farm positive?Are there enough samples from the farm?Were the controls valid?

Internal Control for Detection of False Positive Results

Competitive ICUses the same primer sites as viral targetAI matrix reagent beads - Cepheid

Non-competitiveMultiplex – completely different target and PCR in the same tubeSpiked positive control – duplicate well with diagnostic specimen and spiked +

Background Fluorescence

Is a normal property of Real Time PCRFluorescence derived from unbound probe, free dye, non-specific cleavage of probe or sample auto-fluorescenceRepresents the baseline phase Log-linear phase represents background + fluorescence from amplified DNA

Total FU – background FU = specific FU

Background Subtraction

Corrects for any positive or negative driftCalculates the average background signal and subtracts this from each data pointBetween Bkgnd Min and Max Cycle After a cycle threshold is detected there is no further background subtractionAll calculations are performed and applied individually for each site

Background Fluorescence Represents the Baseline of a Real Time PCR Growth Curve

Raw fluorescence data provides essential information about the magnitude of the background signal and the shape of the growth curve without drift correction.

BackgroundFluorescenceOff

Background Fluorescence On

Background fluorescence is derived from unbound probe•Free dye•Non-specific cleavage of probe•Sample auto-fluorescence

Background Subtraction

Lab Equipment Logistics

Bio-safety cabinet space3 Dedicated cabinets

1. RNA extraction (full exhaust for Trizol® & Qiagen®)2. RNA transfer to reaction tubes (BSC or PCR cabinet)3. Clean reagents, master mix preparation (Cell culture hood, BSC, or PCR cabinet)

If a 2 cabinet system is used RNA transfer and master mix preparation can be conducted in the same hood if the hood is cleaned routinely with 10% bleach solution or Vircon-S

Lab Equipment Logistics

Preparation of clean reagents, extraction and RNA transfer should not be conducted in the same laboratory space as electrophoresis of amplified RNA

PipetsIdeally 3 sets

1. RNA extraction2. RNA transfer3. clean reagents

2 sets – increases possibility of false +1. RNA extraction and transfer2. clean reagents

ONLY USE AEROSOL RESISTANT TIPS

Sample Storage

Swab materials4 C for 3-4 days, more than 4 days (-70 C)

Tissue samples-20 C short term storage, Long term -70 C

Sample RNA4 C less than 24 hrs., more than 24 hrs. -70 C

Control RNA 4 C up to 2 weeks, Long term -70 C (aliquot)

Probe 4 C up to 2 weeks, Long term -20 or -70 C (aliquot)

Avoid multiple freeze thaw cycles for everything

Sample

RNA extraction

APMV-1 MatrixRRT-PCR

Cree/CalMexRRT-PCR

Positive

No further testing

Positive

Report to NVSL for Confirmation with VI and B1 RRT-PCR (vaccine)

Report to NVSL for Confirmation with VI and

RRT-PCR

Negative

Negative

APMV-1 RRT-PCR

Sample

RNA extraction

AIV MatrixRRT-PCR

H5 & H7 RRT-PCR

Positive

No further testing

Positive

Report to NVSL for

Confirmation with VI

Report to NVSL for Confirmation with VI and

RRT-PCR

Negative

Negative

AIV RRT-PCR

Assay Validation

RNA extractionMethods and sample types compared

RRT-PCRPrimer targetsCompared primers setsCompared with VI as “gold standard”

Calculation of Background Subtraction

Bkgnd Min (5) and Max (28) cycle define the range that can be used to calculate the average background fluorescenceThe 4 most recent cycles of data are not included in the calculations to avoid using specific fluorescence data

Calculation of Background Subtraction Continued

At least 5 data points are used to calculate background

Cycles 5,6,7,8,9 when the Bkgnd Min is 5

The bkgnd sub is not applied till cycle 13 1st cycle for detection of positive specimen

Cycles 10,11,12,13 are not included 4 most recent cycle

This occurs until a threshold crossing occurs

APMV-1 RRT-PCR Assay

APMV-1 primer/probe

Target: Matrix gene

Will detect most APMV-1 isolatesVirulent NDVAvirulent vaccine strainsPPMV

Cree/CalMex-VFP-1primer/probeTarget: fusion gene cleavage site

Designed to detect the CA 2002/03 strain of vNDVWill detect most velogens and mesogens.Will not detect vaccine strainsWill detect some PPMV

1. Real-Time RT-PCR using Applied Biosystems® Sequence Detection Systems The following procedures should be used with the Applied Biosystems Sequence Detection instruments (ABI). The following methods were validated with the 7900HT system, and other systems (7000, 7300, 7500) should operate similarly when the 9600 emulation mode is selected. The ABI Sequence Detection System uses an internal passive reference molecule (ROX™), which acts as a normalization factor for fluorescent emissions detected in the samples. The master mix formulas have been adjusted to include a ROX™ reference dye (Catalog # 12223-012, Invitrogen, Carlsbad, CA). THESE MASTER MIX FORMULAS SHOULD ONLY BE USED WITH THE ABI SYSTEMS. THE ROX DYE WILL INTERFERE WITH SMART CYCLER DATA COLLECTION. Table 4. Real-time RT-PCR reaction mix volumes and conditions for type A influenza (MA gene), H5 and H7 primer/probe sets using the ABI Sequence Detection System

The ABI Sequence Detection systems use a 96-well plate format. Before setting up reactions, the PCR plate should be placed into a Splash-free Support Base (P/N 4312063, ABI, Foster City, CA). The base is used to protect the bottom of the plate from picking up particles that may interfere with the optical system. Any residual dust, disinfectant materials, etc. on the bottom of the plate may alter the background fluorescence in that well position.

Volume Per Reaction

Final Concentration

Volume for ___ Reactions

H2O 6.45 μl 5X buffer 5 1X

25mM MgCl2 1.25 3.75 mM* dNTP’s (10 mM each) 0.8 320 μM ea. dNTP

Forward Primer (20 pmol/ul)

0.5 10 pmol/25μl

Reverse Primer (20 pmol/ul)

0.5 10 pmol/25μl

Rnase Inhibitor 13.3 units/µl

0.5 0.266 units/µl

Enzyme Mix 1.0 Probe (6 pmol/ul) 0.5 0.12 μM

ROX reference dye 0.5 MM per rxn

Template Total

17 8

25μl

The arrangement of the reactions on the plate must match the configuration of information on the corresponding plate document. Add 17 µl of master mix to the PCR plate in the Support Base. Touch the tip to the side of the well to draw all of the liquid out of the pipet tip. Add 8 µl of the test sample RNA to the appropriate well using a pipettor designated for RNA transfer. After all of the sample RNA have been added, add 8 µl of positive control to the designated positive control well (using a pipettor designated for transcribed RNA), and 8 µl of RNase free water to the designated negative control well. After all of the RNA have been added to the PCR plate, place an optical adhesive cover (ABI catalog #4311971) over the top of the plate. Be sure to press the adhesive cover firmly against the top of the plate using the MicroAmp Adhesive Seal Applicator (as supplied with Optical Adhesive Cover Starter Kit) so that each well is sealed air-tight. If the adhesive cover is not sealed against the plate, there may be evaporation from the wells and results may be jeopardized. Visually verify that each reaction is positioned at the bottom of its well. If the sample is lying against the side wall of the well, or if there is an air bubble at the bottom of the well, the plate may be centrifuged briefly to position all contents at the bottom. Apply the compression pad that is specific to your particular instrument to the sealed optical plate, and place into the ABI machine.

Thermal cycling Conditions for AIV wet reagent PCR for Cepheid Smart Cycler and Applied Biosystems Inc. (ABI) instrumentation Probe/Primer set Step Time Temp

AIV matrix (Smart Cycler)

45 cycles denaturation 1 sec. 94° C

Annealing* 20 sec. 60° C

AIV matrix (ABI) 45 cycles denaturation 15 sec. 94° C Annealing* 1 min. 60° C

H7 (Smart Cycler) 40 cylces denaturation 1 sec. 94°C Annealing* 20 sec. 58°C

H7 (ABI) 40 cycles denaturation 15 sec. 94°C Annealing* 1 min. 58°C

H5 (Smart Cycler) 40 cycles denaturation 1 sec. 94°C Annealing* 20 sec. 57°C extension 5 sec. 72°C

H5 (ABI) 41 cycles extension 5 sec. 72°C denaturation 1 sec. 94°C Annealing* 20 sec. 57°C

The order of programming is different for ABI and Smart Cycler when using a 3 step PCR procedure. For the ABI, it is necessary to program the 5 sec. extension step first, 1 sec. denaturation step second, and the 20 sec. annealing step third. The fluorescence is acquired during the annealing stage which is the third step. ABI instrumentation can not collect fluorescence during the second step of a 3 step PCR. * The fluorescence is acquired at the annealing step.

2. Setting up Applied Biosystems Sequence Detection System reactions Setting up the reactions Create a new document. Select Assay: Absolute Quantification (Standard Curve); Container: 96 Wells Clear Plate from the drop down menus. Select the appropriate protocol under the Template drop down. The matrix protocols are used for screening specimens. The AIV H5 and AIV H7 primers/probes are used to detect these specific subtypes of AIV. The thermal cycling parameters for each protocol are described in appendix D. Click on Add Detector to create a marker for the absolute quantification probe being used. Click New to create a new detector and assign a name, identify the correct reporter dye and quencher dye, and assign a color for the detector. Click OK. Highlight the Detector and click Copy to Plate Document. Click Done. Using the Ctrl and Shift keys, select individual wells or groups of wells on the plate grid that contain reaction mix. In the well inspector, click the Use check box of the marker you want to add to the selected wells. NOTE: The detectors associated with the marker are automatically applied to the selected wells when the marker is placed in Use. Click on each well position and apply the sample ID to the appropriate well (this may also be done after the instrument completes the run). Note the Passive Reference box defaults to ROX. This refers to the passive reference dye that is added to the master mix. Select the Instrument tab of the plate document. If necessary, check the 9600 Emulation box. (When the 9600 Emulation box is checked, the SDS Software reduces the ramp rate of the 7900HT instrument to match that of the ABI PRISM® 7700 Sequence Detection System instrument.) Change the sample volume to 25 µl. Check to ensure the thermal profile is set to the appropriate thermal cycling parameters for the selected assay.

Select File/Save As and enter a unique run name. Connect to the instrument. Open the tray and place the PCR plate in the instrument. Check to be sure that position A1 on the instrument matches position A1 on the PCR plate. Close the instrument tray. Start the run. 3. Interpretation of Results from ABI Sequence Detection Systems After the run has completed successfully, select Analysis>Analysis Settings from the menu. Select Manual Ct and Automatic Baseline. Then select Analysis>Analyze. The results are displayed in the Results tab. Use the Automatic Baseline option to automatically calculate the placement of the threshold. Visually inspect the placement of the threshold value. The threshold should lie in approximately the center of the linear phase of amplification (refer to Figure 1). The amplification plot of each specimen should also be analyzed individually. Aberrant curves should be viewed in the Multicomponent Pane. The multicomponent illustrates absolute change in emission intensity and the SDS software displays cycle-by-cycle changes in normalized reporter signal (Rn). There are up to five curves in the Multicomponent Pane: the reporter component (FAM), the quencher component (TAMRA), the reference component (ROX), the background component, and the mean squared error (mse). The quencher component may not be present if a Black Hole Quencher (BHQ) is used. Check to be sure that the quencher or reference components do not increase as the FAM component increases. If these dyes increase in fluorescence as the FAM increases, these Ct values should be disregarded and the reaction should be repeated.

Procedure for the Roche LightCycler® 1.2 Real-Time Reverse Transcriptase PCR Instrument for the Detection of Avian Influenza and Avian Paramyxovirus-1 with

Official USDA rRT-PCR Protocol

The following procedure should be used with the Roche LightCycler® 2.0 real-time instrumentation for the detection of avian influenza and avian paramyxovirus-1. The procedure describes the modifications that are required for the implementation of NVSL AVPRO1510 and AVPRO1505. Equivalency validation studies were conducted by NVSL to support the necessary changes in the standard protocols for the use of the LightCycler® 2.0 real-time instrumentation. Equipment and Reagents Non-acetylated Bovine Serum Albumin (BSA). It is essential the BSA be non-acetylated as

acetylated BSA is inhibitory to PCR. Recommended sources and preparation of 5 mg/ml concentration for a final

concentration of 250 µg/ml in 20 μl reaction. New England Biolabs (Ipswich, MA) Catalog # B9001S - 10 mg/ml. Dilute 1 in 2 for 5

mg/ml concentration in RNase free water Ambion (Austin, TX) Catalog # 2616 or 2618 – 50 mg/ml. Dilute 1 in 10 in RNase free

water for 5 mg/ml concentration. LightCycler® 20 μl Capillaries (Roche Catalog Number: 11 909 339 001) LightCycler® Centrifuge Adaptors (in a block) (Roche Catalog Number: 11 909 312 001)

Table 1. Real-time RT-PCR reaction mix volumes using Qiagen One-Step RT-PCR Kit:

* Qiagen (catalog # 210210) buffer already contains 2.5 mM MgCl2 at 1X concentration

Component Volume Per Reaction (μl)

Final Concentration

Water 2.4 5x reaction buffer* 4 1x 25 mM MgCl2 1 3.75 mM Enzyme mix* 0.8 Forward primer 0.5 10 pmol Reverse primer 0.5 10 pmol dNTPs* 0.8 400 mM each Probe 0.5 0.3 μM RNase Inhibitor 0.5 0.33 units/µl BSA (5.0 mg/ml) 1 250 μg/ml MM per reaction 12 RNA Template 8 Total volume 20

RT Step Thermocycling conditions for Qiagen® one-step RT-PCR kit. RT Step 1 cycle 30 min 50°C 15 min 95°C Thermocycling conditions for gene specific probe and primer sets: Probe/Primer set Cycles Step Time Temp AIV Matrix 45 cycles Denaturation 10 sec 94°C Annealing* 20 sec 60°C H5 40 cycles Denaturation 10 sec 94°C Annealing* 20 sec 57°C Extension 5 sec 72°C NDV 40 cycles Denaturation 10 sec 94°C Annealing* 30 sec 56°C Extension 10 sec 72°C

*Fluorescence is collected during the annealing stage.

Programming the LightCycler® Instrument LightCycler® 4.0 Software:

1. Start the LightCycler® 4.0 Software by double-clicking on the LightCycler® 4.0 Software icon on the desktop.

2. In the Login dialog box, type your user name and password. 3. To connect to the database on the local computer, select My Computer in the Log

on to box. 4. Click Login. 5. To program a new protocol, access New Experiment in one of the following ways. If the Front Screen is displayed, click on New Experiment to start a run. Otherwise, click the New button, or select New from the File menu and then New Experiment from the New window, or click on the Run button. 6. In the Setup section of the Programs tab, specify general instrument settings:

a. Default Channel: select the 530 Channel. b. Seek Temperature: 30°C. c. Max. Seek Pos.: enter the number of sample positions the instrument

should look for. d. Instrument Type: choose the 6 Ch. Instrument type for your LightCycler®

2.0 Instrument (this is default). For LightCycler® 1.2 Instrument, select the 3 Ch. Instrument type.

e. Capillary Size: for the 6 Ch. Instrument type, select the capillary size (20 or 100 μl).

5. In the Programs section of the Programs tab, click (+) to add a new program. 6. Edit the default values for the program parameters, clicking the tab button on your

keyboard to move from one column to the next.

Parameter Description/Instructions Valid Values Program Name The name for the program. Click in the

Program Name box, then enter a new name. Any alphanumeric string

Cycle The number of times the program should be repeated. Enter a value or select it by clicking on the up and down arrows.

1-99 cycles

Analysis Mode The type of analysis expected for this program (if any). Select an analysis mode from the pull-down list.

None: no analysis Melting Curves: a melting curve analysis is expected. Quantification: a quantification analysis is expected. Color Compensation: a color compensation analysis is expected.

7. In the Temperature Targets section, edit the default values for the temperature

parameters. Parameter Description/Instructions Valid Values Value of

AI Matrix RT

Value for AI Matrix PCR assay

Target The target temperature. Enter a temperature.

37°C-98°C 50 step 1 94 step 2

94°C

Hold The length of time to hold the target temperature in hours:minutes:seconds. Enter a hold time.

00:00:00-12:00:00 30:00 step 1 15:00 step 2

1.0

Slope The speed with which the temperature must be reached, specified in degrees per second. Enter a slope.

0.05°C - 20°C per second.

20.0 20.0

Sec. Target A second target temperature to be reached by the last cycle of the program. Useful for Touchdown PCR. Enter a temperature.

37°C-98°C 0.00 0.0

Step Size The number of degrees to change the temperature with each cycle to reach the secondary target. Enter a step size.

0°C - 20°C 0.00 0.0

Step Delay The cycle number at which the temperature step up or step down begins. Enter a cycle number.

0-99 cycles 0.00 0.0

Acquisition Mode

The frequency with which fluorescence data is acquired. Select an acquisition mode from the pull-down list.

None: no fluorescence data is acquired. Single: acquires fluorescence data once at the end of this temperature segment in each cycle. Continuous: acquires fluorescence data continuously. Step: acquires fluorescence data at each temperature transition.

None Single

8. Click (+) to add another temperature target to the current program, then enter

parameter values. Repeat to define as many temperature targets, as you need for the current program.

9. Repeat steps 7 – 10 to create additional programs and their temperature targets. Programs or temperature targets can be reordered by selecting the item you want to move, then clicking the up or down arrow. To delete an item, select the item, then click (-).

10. Look at the Overview section to see a graphical representation of all the programs you have defined.

11. Click Save in the global toolbar to save the protocol. Navigate to a location to save the protocol, enter a protocol name, then click OK.

LightCycler® 3.5.3 Software:

1. Start the LightCycler® 3.5.3 Software by double-clicking on the LightCycler® 3 Front Screen icon on the desktop. Alternatively, in the Windows Start Menu bar, click on the LightCycler® Front Screen under the LightCycler® 3 folder.

2. In the LightCycler® Front Screen, click the Run button to enter the Programming Screen. If you have not switched on the LightCycler® instrument, the software will prompt you to do so.

3. A dialog box will appear which offers the execution of an optional 1-2 minutes self test. The performance of one self test a day is recommended.

4. In the programming screen, click on the New Experiment button to create a new Experimental Protocol. To open and modify an existing file, select the Open Experiment File button.

5. Use the buttons in the Cycle Program Field to define the Cycle Programs for your Experimental Protocol:

Button Function Add Creates a new Cycle Program. Remove Removes a selected program. Import Imports a cycle program from other experiment protocols. Move Up Changes the order in which cycle programs will be executed.

6. To alter the name of a Cycle Program, double click on the name in the Cycle Program field. A window will pop up which allows change of the name.

7. Upon addition of a Cycle Program, the Cycle Program Data field in the middle of the screen is activated.

8. Use the Temperature Targets Segment to define the temperature profiles for each individual program: Click on the green Ins button to enter a new temperature segment. Click on the red Del button to delete a temperature target.

9. Enter the appropriate target temperatures and times: Field Purpose Target Temperature (°C) Defines the temperature of the segment in °C. Incubation Time (h:min:secs)

Defines the holding time of a temperature segment.

Temperature Transition Defines rates at which the instrument changes temperature between temperature targets. The slowest rate is 0.1°C/sec and the fastest rate is

Rate (°C/sec) 20°C/sec. Secondary Target Temperature (°C)

Defines a second target temperature within a segment beginning at a defined cycle number. This is for Touchdown PCR.

Step Size (°C) Defines the degree of change per cycle used to step up/step down from the Target Temperature to the Secondary Target Temperature.

Step Delay (cycles) Defines the cycle number at which the step up/step down from the Target Temperature to the Secondary Target Temperature begins.

10. Specify the fluorescence acquisition mode:

Type Description None No fluorescence measurement. Single Fluorescence is measured once per sample at the end of the temperature

segment selected. Cont. (Continuous) Fluorescence of all samples is measured continuously from the first sample to

the last one. Step (Stepwise) Fluorescence of all samples is measured after each temperature transition.

11. Select the correct Analysis Mode for the Cycle Program: a. Default setting is None (no data analysis is intended for this cycle

program) b. Choose Quantification for later quantification analysis of the data. c. Choose Melting Curve Analysis for later analysis of melting curve data.

12. Type in the number of cycles to be run with the selected Temperature Targets. The resulting Cycle Program profile can be monitored in the Cycle Simulation field.

13. Add all Cycle Programs needed for the Experimental Protocol. The resulting Experimental Protocol profile can be monitored in the Experiment Simulation field.

14. Select the Fluorescence Display Mode according to the detection system you have chosen.

15. Click the Save Experiment File button to save a newly defined or modified Experimental Protocol. The file is automatically saved as *.exp in the User directory.

Setting Up Roche LightCycler® Reactions

1. In a 1.5 ml tube, prepare reagent mastermix according to table 1(everything except RNA template) as described above. To prepare the mastermix for more than one reaction, multiply the amount in the “Volume per reaction” column by z, where z = the number of reactions to be run plus 1-2 additional reactions (compensates for pipetting errors). To eliminate laboratory contamination it is necessary to prepare the reagent mix in a dedicated clean reagent hood with pipettes and filtered pipette tips that are dedicated to the preparation of clean reagents.

2. Place the number of LightCycler® capillaries required in the pre-cooled LightCycler® Centrifuge Adaptor Block.

3. Pipette 12 microliters of mastermix into each capillary. 4. Pipette 8 microliters of RNA (controls or unknown samples) into each capillary.

The transfer of RNA should be conducted in a PCR workstation or biosafety cabinet that is not used for the extraction of RNA from diagnostic specimens or the preparation or clean reagents.

5. Cap each capillary with the supplied caps using the LightCycler® Capping Tool. When capping make sure to press straight down on capillary, not from an angle. Lift capped capillary out of adaptor and release cap from Capping Tool.

6. If you have a LightCycler® Carousel Centrifuge, place capillaries into the LightCycler® Carousel and centrifuge according to the manufacturer’s instructions, prior to placing the LightCycler® Carousel in the LightCycler® instrument. If you do not have a LightCycler® Carousel Centrifuge, centrifuge LightCycler® capillary adaptors with capped capillaries in a microcentrifuge. Only a short pulse is required. Then place centrifuged capillaries in the LightCycler® Carousel, prior to placing the loaded carousel in the LightCycler® instrument.

7. Set up the cycling parameters as described above and start the RT-PCR by clicking on the “Start Run” button.

Interpretation of Results from Roche LightCycler® Detection Systems using the Automated Method LightCycler® 4.0 Software: The automated absolute quantification method uses a different algorithm method of calculating the crossing point (CP) than the Fit Points method. Equivalency testing has not been conducted on the Fits Points method. The Fits Points method should not be used to interpret and analyze results for NVSL AVPRO1510 and AVPRO1505.

1. Click Analysis on the main toolbar. 2. Select Absolute Quantification, then click OK 3. In the Sample Editor, enter specimen identification information on the Capillary

View tab, and select the channel (FAM) to be used in the experiment. 4. On the Abs Quant tab of the Sample Editor, enter sample information as follows:

Column Name Valid Values Description Target Name Any name Name of the target for this

channel Type Unknown Standard Type of sample in this

capillary Concentration Any concentration value Concentration of a standard

sample

5. Click Abs Quant in the module bar to open the analysis module. 6. From the Channel menu select the channel (FAM) for the targets you want to

analyze. 7. To see the crossing points and concentrations, drag or click on the slide bar.

LightCycler® 3.5.3 Software:

1. In the LightCycler® Front Screen, click on the Analysis button. 2. Select your experiment and click the Open button. Alternatively double-click your

experiment. 3. Select the appropriate part of the run to be analyzed in the Select a Program drop

down menu: Select Cycles. 4. Adjust the y-axis for the Fluorescence graph: Choose F1 axis setting for TaqMan

Probes. 5. Select the type of analysis you want to perform:

a. Click on Quantification to proceed to the Quantification Screen. 6. Highlight the samples to be analyzed. 7. Select Second Derivative Maximum in the Analysis field (top left corner). 8. Click Proportional in the Baseline Adjustment box (to the right of the Analysis

box). 9. Then select Step 2: Analysis to see the standard curve. 10. Crossing points and concentrations will be displayed next to the sample names. 11. If sample names and concentrations of standards need to be edited after the run:

a. From the LightCycler® Front Screen, click on Options and select LC Data File Editor.

b. Click the Open button and find and select your experiment. Click the Open button, or double-click the experiment to open it.

c. Click on the Change Samples button to enter the Sample Editor. d. Edit the names and/or concentrations and click on the Done button. e. Click the Save button to save the changes. f. Click the Exit button to exit the data editor.

Tips for preventing breakage of capillaries:

1. Handle the capillaries with care; prevent breakages by not “bending” capillaries. 2. When capping make sure to press straight down on capillary, not from an angle.

Lift capped capillary out of adaptor and release cap from Capping Tool. 3. Before loading capillaries in the LightCycler® Carousel, clean out the carousel

with the dental floss cleaner. 4. If a capillary breaks in the carousel, make sure that there is no glass shards left in

the hole by cleaning with the dental floss cleaner. 5. Replace the rubber ring in the carousel before the rubber starts to break down and

become hard.

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Prepare the reaction master mix in a 1.5ml tube

6.0Rnase Inhibitor6.0Probe9.6dNTP’s6.0Reverse Primer6.0Forward Primer12Enzyme Mix

15.025mM MgCl2

605X83.4H2O

Volume in μlComponent

7.0Rnase Inhibitor7.0Probe11.2dNTP’s7.0Reverse Primer7.0Forward Primer14.0Enzyme Mix17.525mM MgCl2

705X97.3H2O

Volume in μlComponent

4 people/group 5 people/group

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