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Contents lists available at ScienceDirect

Journal of Clinical Virology

journa l homepage: www.e lsev ier .com/ locate / j cv

P5+/6+ SYBR Green methodology for simultaneous screening and quantificationf human papillomavirus

elissa Rodrigues de Araujoa,b, Laura De Marcob, Carlos F. Santosa, Izabel Regina Fisher Rubira-Bullena,uglielmo Roncoc, Ilenia Penninib, Loredana Vizzinib, Franco Merlettib,c, Anna Gillio-Tosb,∗

Bauru School of Dentistry, University of São Paulo, Al. Octávio Pinheiro Brisola, 9-75, Bauru, São Paulo 17012-901, BrazilUnit of Cancer Epidemiology, C.E.R.M.S., University of Turin, Via Santena 7, 10126 Turin, ItalyCenter for Oncologic Prevention, Via San Francesco da Paola 31, 10123 Turin, Italy

r t i c l e i n f o

rticle history:eceived 17 November 2008eceived in revised form 9 March 2009ccepted 19 March 2009

eywords:uman papillomaviruscreeningiral loadYBR Greeneal-time PCR

a b s t r a c t

Background: Detection and quantification of human papillomavirus (HPV) may help in predicting theevolution of HPV infection and progression of associated lesions.Objectives: We propose a novel protocol using consensus primers GP5+/6+ in a SYBR Green quantitativereal-time (Q-RT) polymerase chain reaction (PCR). The strategy permits screening for HPV infection andviral load quantification simultaneously.Study design: DNA from 153 archived cervical samples, previously tested for HPV detection by GP5+/6+ PCRand typed by EIA-RLB (enzyme immunoassay-reverse line blot) or sequence analysis, was analysed usingSYBR Green Q-RT PCR. Melting temperature assay (Tm) and cycle threshold (Ct) were used to evaluate HPVpositivity and viral load. The Tm in the range of 77–82 ◦C was considered to be positive for HPV-DNA. HPVresults generated through GP5+/6+ conventional PCR were considered the gold standard against whichsensitivity and specificity of our assay were measured.

Results: Out of 104 HPV positive samples, 100 (96.2%) were also determined as positive by SYBR GreenQ-RT PCR; of the 49 HPV-negative samples, all were determined as negative. There was an excellentpositivity agreement (� = 0.94) between the SYBR Green Q-RT and the previous methods employed. Thespecificity and sensitivity were 100% and 96.2%, respectively. Comparison of SYBR Green Q-RT and TaqManoligo-probe technologies gave an excellent concordance (�c = 0.95) which validated the proposed strategy.Conclusions: We propose a sensitive and easy-to-perform technique for HPV screening and viral loadquantification simultaneously.

. Background

Human papillomaviruses (HPVs) are recognised as risk factorsor development of skin and mucous membrane neoplasias.1–5 Thedentification of HPV infection alone may not represent a sufficientuideline to identify patients at increased risk for developing can-er. Several viral factors may play a role in carcinogenesis, includingPV genotype, multiple infections, viral load, HPV variants andiral integration.3,6 A conclusive agreement has thus far not beenchieved regarding the value of these parameters, which are still

Please cite this article in press as: de Araujo MR, et al. GP5+/6+ SYBR Gof human papillomavirus. J Clin Virol (2009), doi:10.1016/j.jcv.2009.03

nder investigation to evaluate their predictive power.7 In the lastecade, attention to viral load relevance has increased becauseigh viral load has been associated with persistent infection androgression to high-grade lesions.8–10 Although the utility of viral

∗ Corresponding author. Tel.: +39 011 6336863; fax: +39 011 6334664.E-mail address: gilliotos demarco@yahoo.it (A. Gillio-Tos).

386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jcv.2009.03.020

© 2009 Elsevier B.V. All rights reserved.

load varies with HPV type and a clinical application should beapproached with caution,10 some authors claim that viral load per-mits identification of high-risk populations regardless of the levelof lesion severity, thereby allowing selection of more appropriatemanagement protocols.9,11,12

Determination of the HPV titre may help when assessing thepossible evolution of an infection towards clearance or lesion pro-gression. Reduction of viral load may predict the regression ofcervical intra-epithelial neoplasia to normality.13

Several methods for HPV quantification have been described;however, most of them use TaqMan oligo-probes technology andare mainly focused on HPV16, 18, 31 and 33.14 These methodsare expensive, time consuming and are not available for all HPV

reen methodology for simultaneous screening and quantification.020

types. Indeed, few studies described the quantification for othergenotypes.13,15 The diversity of HPV genotypes and high incidenceof multiple infections require to establish reliable methods for HPVidentification and quantification for epidemiological studies andpotentially for patient management.16

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Table 1HPV positivity using GP5+/6+ EIA-RLB or direct sequence analysis genotypes andGP5+/6+ SYBR Green analysis.

Number of samples GP5+/6+ EIA-RLB ordirect sequence analysis

GP5+/6+ SYBR Green

Single infections Positive Negative

HPV low-risk type2 6 2 01 11 1 01 42 1 03 70 3 04 81 4 02 90 2 07 Others (32,43,54, 72, 74,

87, 89)7 0

HPV high-risk type24 16 24 0

4 18 4 04 31 4 01 33 1 03 39 2 14 45 4 03 51 3 04 56 4 04 58 4 03 59 3 03 66 3 03 67 3 02 68 1 14 Others (52, 73, 82, 91) 4 0

Number of samples GP5+/6+ EIA-RLB ordirect sequence analysis

GP5+/6+ SYBR Green

Multiple infections Positive Negative

14 Double infectiona 12 22 Triple infectionb 2 0

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. Objectives

The objective of this study was to perform a novel protocolmploying the quantitative ability and dynamic range providedy real-time polymerase chain reaction (PCR) in an SYBR Greenethodology using the consensus primers GP5+/6+. These primers

re known for their efficient detection of a broad spectrum of HPVenotypes associated with neoplastic evolution.17 We propose atrategy that permits to combine the screening for HPV infectionnd the quantification of HPV viral load by using a low-cost fluo-ophore and quick microplate setting.

. Study design

.1. Study population

A total of 153 archived DNA samples positive and negative forPV detection from the previous studies18,19 were used.

DNA had been extracted from cervical samples obtained fromomen aged 25–70 years attending a screening programme for cer-

ical cancer in Turin, Italy. As described previously,18,19 DNA hadeen purified from 100 �l of cervical cell samples which presentedmean of 5.25 × 108 cells per 100 �l (range: 3.3 × 102 to 4.97 × 1010

ells per 100 �l) by silica-gel columns (Qiagen, Milan, Italy). All DNAamples had been tested for HPV positivity by PCR analysis employ-ng GP5+/6+ primers. The positive samples had subsequently beenenotyped by GP5+/6+ EIA-RLB (enzyme immunoassay-reverse linelot) or, when not identified by this method, by direct sequencenalysis. Residual material had been stored at −80 ◦C.

All DNA samples before inclusion in this study were evaluated bymplification of the �-globin gene as an internal control for DNAdequacy, to avoid false-negative results in the quantitative real-ime (Q-RT) PCR assays.

The sample distribution is as follows: 104 were positive for HPV84 high and 20 low risk) according to the IARC classification,1 and9 were negative (Table 1).

.2. Cell lines

SiHa20 and CaSki21 cervical carcinoma cell lines harbouringPV16 genomes (ATCC, Manassas, VA, USA), were used as controls

or cellular HPV-DNA. DNA isolation from cell lines was performedy silica gel columns (Qiagen, Milan, Italy).

.3. SYBR Green quantitative real-time (Q-RT) PCR

Quantification of the �-globin gene and HPV-DNA were carriedut in an iCycler iQTM Real-Time PCR Detection System (BioRad®,ercules, CA, USA).

Samples and controls were analysed in duplicate.22 NegativeDNA-free) and positive controls were included in each run.

All reactions were performed in a final volume of 25 �l, contain-ng 2× SYBR Green Supermix (BioRad®), either 0.3 �M of �-globin23

r 50 pmol of each GP5+/6+16 primers, and 1 �l of DNA sample.The amplification protocol for �-globin analysis was: 90 s at

5 ◦C, 50 repeats at 95 ◦C for 15 s and 50 ◦C for 1 min. After ampli-cation, a melting curve was performed at temperatures between5 ◦C and 95 ◦C, with temperature increasing at a rate of 0.5 ◦C s−1.

For HPV-DNA analysis the amplification protocol was optimisedefore testing the study samples. The annealing temperature was

Please cite this article in press as: de Araujo MR, et al. GP5+/6+ SYBR Gof human papillomavirus. J Clin Virol (2009), doi:10.1016/j.jcv.2009.03

elected by automatic gradient analysis in the range between0.0 ◦C24 and 38.0 ◦C25,26 by testing six HPV-DNA negative, fourPV-DNA positive (types 18, 54, 66, 82), one CaSki DNA, one SiHaNA and one DNA-free samples. HPV-DNA positive samples (cell

ine included) chosen for the optimisation of the protocol had a phy-

a Double infections genotypes: 16/18, 16/31, 16/56, 16/59, 16/66, 18/51, 18/58,31/39, 31/56, 35/53, 39/66, 56/58, 56/68, 66/68;

b Triple infection genotypes: 16/33/56, 16/56/82.

logenetic heterogeneity, since they belong to five different speciesclades (7, 13, 6, 5 and 9).

To verify the correct discrimination between GP5+/6+ ampliconsand non-specific or primer dimer products as well as PCR efficacy,gel electrophoresis was performed onto a 2% agarose gel.27 A 48.0 ◦Cannealing temperature produced the best yield and discrimina-tion of the target. Lower temperatures showed lack of specificity.Higher temperatures decreased the sensitivity. Therefore, we usedthe following amplification protocol for HPV-DNA analysis: 90 s at95 ◦C, followed by 50 repeats at 94 ◦C for 15 s and 48 ◦C for 30 s.After amplification, a melting curve was performed at tempera-tures between 65 ◦C and 95 ◦C, with temperature increasing at arate of 0.5 ◦C s−1. We identified the melting temperature (Tm) rangeto discriminate GP5+/6+ amplicons between 77 ◦C and 82 ◦C.

External standard calibration curves were generated using10-fold dilutions (2 × 102 ng �l−1 to 2 × 10−1 ng �l−1) of humanplacental DNA (Sigma–Aldrich, St. Louis, MO, USA) or 10-fold dilu-tions (106, 105, 104, 103 and 102 viral copies) of a plasmid carryingthe complete HPV16 genome (Clonit, Milan, Italy). The Ct (cyclethreshold) values were calculated with a software program fordata analysis using an automatic setting of the baseline (BioRad).The �-globin amounts were given as nanogram of �-globin in 1 �lof input DNA. Viral load results were reported as HPV copies/cellequivalent.28 The sensitivity of the method was 102 viral copies.

reen methodology for simultaneous screening and quantification.020

3.4. TaqMan Q-RT

TaqMan Q-RT assays were performed using primers and TaqManoligo-probes previously described that target the �-globin gene23

and HPV16 E6 region,29,30 respectively. Amplification conditions

Please cite this article in press as: de Araujo MR, et al. GP5+/6+ SYBR Green methodology for simultaneous screening and quantificationof human papillomavirus. J Clin Virol (2009), doi:10.1016/j.jcv.2009.03.020

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Fig. 1. Example of an amplification plot, standard curve, and melting curve for HPV detection by GP5+/6+ SYBR Green Q-RT PCR. (a) Amplification plot of GP5+/6+ SYBR GreenQ-RT PCR. (b) Q-RT PCR standard curve. (c) Melting curve assay showing HPV positive samples: (*) HPV positive samples, Tm 77 ◦C; (§) HPV16 positive controls, Tm 79 ◦C;(∞) HPV positive samples, Tm 81.5 ◦C. (d) Melting curve assay showing HPV negative samples: (§) HPV16 positive controls, Tm 79 ◦C; (†) negative samples, Tm 70 ◦C, primerdimers; (�) negative samples, not amplified, no Tm.

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orophores. The HPV16 positive DNA samples (N = 24), along withCaSki and SiHa DNA, were tested with both the technologies. Anal-ogously, the sample DNA amount was evaluated using the �-globinprimers and SYBR Green or a �-globin oligo-probe.23 Fig. 3 shows

Fig. 3. Comparison between GP5+/6+ SYBR Green Q-RT and TaqMan oligo-probe

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ere: 10 min at 95 ◦C, 50 repeats for 15 s at 95 ◦C and 1 min at 50 ◦Cr 57 ◦C for �-globin gene or HPV16 E6 assays, respectively.

External calibration curves were generated and the Ct valuesutomatically calculated as described above. The sensitivity of theethod was 102 viral copies.HPV viral load results were reported as HPV copies/cell

quivalent.28

.5. Statistical analysis

Statistical analyses were performed by using StataCorp soft-are (Stata Statistical Software Release 10, College Station, TX, USA:

tataCorp LP, 2007). The GP5+/6+ EIA-RLB test17,18 was taken as aeference. Kappa statistics31 were used to assess the concordanceetween pair-wise methods in HPV positivity.

The diagnostic accuracy parameters were calculated by sensitiv-ty and specificity calculations, expressed as percentages with 95%onfidence intervals (CIs).

The concordance correlation coefficient (�c), containing a mea-urement of precision and accuracy,32 was used to evaluate thegreement between paired methods in HPV viral load determina-ion.

. Results

The system we proposed was designed to allow simultane-us screening and quantification of all HPV genotypes that can beetected by GP5+/6+ primers.

.1. HPV screening

The results are summarised in Table 1.To discriminate HPV positivity, we used the Ct value (Fig. 1a and

) combined with the Tm22,27,33 We found our HPV16 positive con-

rols showing a Tm of 79 ◦C, according to previous demonstrations.24

Tm in the range of 77–82 ◦C was considered to be positive forPV-DNA detection by GP5+/6+ (Fig. 1c and d), since all ampliconsith a Tm in this range showed a sharp band of the correct size of

50 bp when gel electrophoresis was performed. Deviations fromhe range indicated either a non-specific product or primer-dimer

elting signals, which were confirmed by gel electrophoresis. Thus,e adopted the following criteria for the evaluation of HPV positiv-

ty of the samples: the Tm must be in the range mentioned aboveith a sharp, high resultant peak resembling the standard curve ref-

rence in terms of the ratio of its height and width. Samples withouteaks or showing flat peaks and a Tm out of the range were identi-ed as negative for HPV. Primer dimers as well as non-specific PCR

ragments showed a different Tm (around 70 ◦C) than expected27

nd a Ct out of the range established by the standard curve (Fig. 1d).We found excellent positivity agreement between screening for

PV with our SYBR Green Q-RT PCR and the original screeningethods18,19 (� = 0.94, 95% CI = 0.88–0.99). Out of the 104 HPV sam-

les classified as positive by GP5+/GP6+ EIA-RLB, 100 were alsoetermined to be positive by SYBR Green Q-RT PCR; all 49 HPV-egative samples were determined to be negative by our method.herefore, the sensitivity and specificity in our results were 96.2%95% CI = 92.5–99.8%) and 100%, respectively.

Sixteen multiple infections were included in the analysis. Four-een of these (87.5%) were identified as positive by our systemTable 1).

Please cite this article in press as: de Araujo MR, et al. GP5+/6+ SYBR Gof human papillomavirus. J Clin Virol (2009), doi:10.1016/j.jcv.2009.03

.2. HPV viral load

Viral loads from HPV positive samples evaluated by SYBR Green-RT PCR are shown in Fig. 2. The viral copy number, normalised to

he input DNA for each sample with the �-globin assay and reported

Viral load results are reported as Log10 HPV copies/cell equivalent. Samples weregrouped based on the positivity to HPV16, HPV18, HPV31, HPV HR (high-risk types,different from HPV16, 18, 31), HPV LR (low-risk types), and HPV Mixed (samplesharbouring multiple infections).

as HPV copies/cell equivalent, varied from 3.86 × 10−4 to 1.28 × 105.For HPV16 positive samples with a single infection (N = 24), themedian viral load was 2.37 × 101 HPV copies/cell equivalent. Addi-tionally, the amounts at the 25th and 75th percentiles were 3.92and 1.37 × 102, respectively.

4.3. Method validation

Validation of our SYBR Green Q-RT PCR for HPV quantificationwas performed by comparison with TaqMan Q-RT technology. Thecomparison was restricted to detection of the HPV16 E6 regionusing HPV16 E6 primers and SYBR Green or E6 oligo-probe29,30 flu-

reen methodology for simultaneous screening and quantification.020

Q-RT assays. The analysis included the 24 HPV16 positive DNA samples along withCaSki and SiHa DNA. Paired viral load results for the samples tested with both thetechnologies were reported as Log10 HPV copies/cell equivalent: (♦) SYBR Green Q-RT assay; (�) TaqMan oligo-probe Q-RT assay. In two cases (*), the viral load valueswere almost equivalent; therefore, only one symbol is visualised, while the other ishidden.

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he HPV16 copies/cell equivalent obtained with the two technolo-ies, demonstrating an excellent concordance (�c = 0.982, 95% CI:.921–0.983) between the two methods.

. Conclusions

There is increasing interest in the use of quantification methodso better predict the evolution of HPV infection. Although a ‘clini-al significance’ of HPV viral load and its prognostic value are stillnder investigation,10 viral load of HPV-DNA has been proposeds a biomarker of persistent infection,15 which might lead to anncreased occurrence of cervical lesions.34

The present study describes a suitable real-time PCR for per-orming HPV screening and quantification simultaneously, thereby

inimising the number of parallel analyses for each sample. The usef the widely described GP5+/6+ primers permits the evaluation ofiral load of different HPV types (including multiple infections) inhe same set of analyses for HPV screening, although identificationf high- and low-risk subtypes will be necessary in the follow-upor clinical management of HPV infection. SYBR Green was cho-en as a fluorophore that intercalates with double-stranded DNAnd may determine the total HPV viral load without suffering fromemplate-sequence variation among types.

Small sample sizes and use of archived samples with incompleteathological information can be listed as limitations of the study. Toscribe clinical sensitivity/specificity of the assay, we plan furtheralidation on a greater number of samples, by testing longitudinaleries and measuring disease outcomes.

Despite limitations of the study, our methodology showed highnalytical sensitivity, as demonstrated by the good agreementound in the samples tested between the detection of HPV byP5+/6+ SYBR Green Q-RT and the original analysis by GP5+/6+IA-RLB or direct sequencing analysis.

Only four (3.85%) discrepant results between these methodsere observed, and all were false negative (Table 1). These discrep-

ncies may be due to a low HPV viral load in the sample, whichould cause deviations in Tm towards non-specific peaks as pre-iously reported.35 No false-positive results were obtained usinghe SYBR Green methodology, which demonstrates the specificitychieved. The reliability of the viral load estimated by GP5+/6+ SYBRreen Q-RT was also supported by comparable viral load valuesbtained when HPV16 positive samples were analysed with bothYBR Green Q-RT and TaqMan oligo-probe technologies. Indeed, theomparison resulted in an excellent concordance between theseests, showing the detection of amplified product with approxi-

ately the same sensitivity as that described by other authors.6

HPV-DNA quantification is currently established for a few HPVenotypes.14 Measuring viral loads in other HPV genotypes maye relevant for managing high-risk subsets of populations,15 and aethodology using the easy-to-handle22 and low-cost SYBR Greenay be convenient.In conclusion, this study described a sensitive technique for the

creening and quantification of a wide range of HPV-DNA that isuitable for large-scale studies37 of high-risk patients, and is inex-ensive and easy to perform.

onflict of interest

None of the authors has competing interests.

Please cite this article in press as: de Araujo MR, et al. GP5+/6+ SYBR Gof human papillomavirus. J Clin Virol (2009), doi:10.1016/j.jcv.2009.03

cknowledgments

M.R. Araujo, received a PhD scholarship from CAPES, Brazilianovernment (2184-07-9).

PRESSal Virology xxx (2009) xxx–xxx 5

This work was partially supported by the Special Project ‘Oncol-ogy’, Compagnia di San Paolo/FIRMS, the Italian Association forCancer Research and by Piedmont Region.

Ethical approval was not required for this study.

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