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Journal of Virological Methods 196 (2014) 45– 49

Contents lists available at ScienceDirect

Journal of Virological Methods

jou rn al hom ep age: www.elsev ier .com/ locate / jv i romet

ncogenic human papillomavirus genotyping by multiplex PCR andragment analysis

iatou Souhoa,b, Bahia Bennania,c,∗

Laboratoire de Microbiologie et Biologie moléculaire, Faculté de Médecine et de Pharmacie de Fès (FMPF), Université Sidi Mohammed Ben Abdellah,ès (USMBA), MoroccoLaboratoire de Biotechnologies Faculté des Sciences Dhar El Mehraz Fès, USMBA, MoroccoEquipe Micro-Organismes et Facteurs Oncogènes, Laboratoire de Biologie des Cancers, FMPF, USMBA, Morocco

rticle history:eceived 28 May 2013eceived in revised form 14 October 2013ccepted 18 October 2013vailable online xxx

eywords:PV

a b s t r a c t

Human papillomavirus (HPV) detection and genotyping are determinant in cervical cancer prevention.They help to identify women at risk and allow the establishment of epidemiologic profiles. Many PCR-based genotyping methods have been developed. They require many steps or specialized equipmentincreasing the assay duration and cost. This affects their routine use, especially in developing countries.Therefore, the aim of this study was to develop a new HPV genotyping method that can be routinely usedalso in low incomes countries. For this purpose, fifteen high risk HPV type specific reverse primers weredesigned on L1 gene and fluorescently labeled. These primers were used on two multiplex PCR with one

ultiplex PCRPV genotypingragment analysisapillary electrophoresis

common forward primer (MY11). The lengths of products were revealed by capillary electrophoresis.This technique identifies sixteen high risk HPVs (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 68,73 and 82). It was optimized on HPV genome plasmids and evaluated on artificial and cervical samples.All the sixteen targeted genotypes were identified specifically and repeatedly in simple and multipleinfections in both artificial and clinical samples. The developed technique is sensitive, specific, easy toperform and appropriate for routine laboratory use and high throughput screening programs.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

Cervical cancer is the second most common malignant diseasen women. It is responsible for high rates of death and affects

ore frequently women in developing countries (American Cancerociety, 2011; WHO, 2002). This cancer has been associated to per-istent infection with high risk human papillomavirus (HR-HPV)Walboomers et al., 1999; McLaughlin-Drubin and Münger, 2009).

In all cervical cancer prevention strategies, HR-HPV detectionnd genotyping are critical and determinant steps. They permit thedentification of persons at risk and patients who require follow-p or treatment. They also give an idea on types distribution andllow the assessment of vaccination efficiency (Scarinci et al., 2010).

mong numbers of assays described for HPV diagnosis, molecularpproach based on viral DNA detection offers the most significantesults. These methods present variable rates of sensibilities and up

∗ Corresponding author at: Laboratoire de Microbiologie et Biologie moléculaire,aculté de Médecine et de Pharmacie de Fès (FMPF), Université Sidi Mohammed Benbdellah, Fès (USMBA), Morocco. Tel.: +212 6 61 73 07 63.

E-mail address: bahia bc@yahoo.fr (B. Bennani).

166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jviromet.2013.10.024

to now, none of them is considered gold standard (Zaravinos et al.,2009).

The most widely used methods of HPV detection target theL1 open reading frame which codes for the major capsid pro-tein. PCR-based detection methods use consensus or degeneratedprimer sets, like GP5+/6+, SPF10, MY11/09 or PGMY11/09 (Gravittet al., 2000; Zaravinos et al., 2009; Poljak et al., 2012). The E6/E7region that codes for oncoproteins E6 and E7 is also targeted bysome genotyping methods (Molden et al., 2007; Zaravinos et al.,2009). HPV genotyping methods are based on molecular detectiontechniques associated with an identification step. This step couldbe DNA sequencing or hybridization with specific labeled probes(Kleter et al., 1999; Digene Corporation, 2004; Castle et al., 2008;Satra et al., 2009; Geraets et al., 2011). Identification steps requiremany and long lasting reactions and sometimes dedicated appara-tus increasing the reaction’s cost.

DNA fragment analysis with capillary electrophoresis appears asan effective method to identify amplified DNA fragments. It offers

a good specificity of detection and requires very low amount oflabeled DNA. It has been used to type many organisms such asEnterococci, Mycobacteria or HPV (Ho et al., 2004; Burtscher et al.,2006; Santiago et al., 2006; Dictor and Warenholt, 2011).

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In this paper, we describe a new HPV genotyping method basedn multiplex PCR and detection by fragment analysis on DNAnalyzer.

. Materials and methods

.1. Primers design

HR-HPV type specific primers were designed on the basis of aultiple sequence alignment which was performed with Clustal

software. The sequences of L1 gene delimited by MY11/MY09rimers and corresponding to 39 HPV genotypes were aligned. Gen-ank accession numbers of the included HR-HPV types sequencesere: HQ644299 for HPV16; GQ180792 for HPV18; HQ537686 forPV31; HQ537705 for HPV33; JX129485 for HPV35; U45903 forPV39; EF202167 for HPV45; M62877 for HPV51; HQ537749 forPV52; EF546475 for HPV53; EF177181 for HPV56; HQ537764

or HPV58; X77858 for HPV59; EU918769 for HPV68; X94165 forPV73; AB027021 for HPV82. For low risk types, included acces-

ion numbers were: HE962031 for HPV6; JQ773412 for HPV11;Q344807 for HPV13; X74474 for HPV30; AB436167 for HPV34;E793059 for HPV40; HE820130 for HPV42; AJ620205 for HPV43;E963129 for HPV44; NC 001676 for HPV54; HE963161 for HPV55;12500 for HPV61; AY395706 for HPV62; U12495 for HPV64;12492 for HPV67; U12497 for HPV69; EF626587 for HPV70;94164 for HPV72; U40822 for HPV74; GQ288790 for HPV81;F151983 for HPV83; AF293960 for HPV84 and AF419318 forPV91.

For each HR-HPV genotype, a specific reverse primer was chosenn the less conserved region of targeted sequence. Thereby, fifteenifferent primers were designed and labeled at the 5′ end (Table 1).our fluorescent dyes (FAM, NED, PET or VIC) were used to labelhese primers in such a way to differentiate sequences with similarizes. The consensus MY11 primer was chosen as unique forwardrimer.

.2. HPV DNA and samples

HPV types 6, 11, 16, 18, 45, 51, 52 and 53 DNA plasmids wereindly provided by Prof. Dr. E.M. de Villiers from German Can-er Research Center (DKFZ). HPV 33, 39 and 68 plasmids wereindly provided by Pr. Michel Favre from “Institut Pasteur” FranceBeaudenon et al., 1986, 1987; Longuet et al., 1996). HPV 31, 35nd 56 plasmids were kindly provided by QIAGEN Gaithersburg,nc. and Dr Attila Lorincz. HPV 58 DNA was obtained from cer-ical sample by MY11/MY09 amplification and sequencing in ouraboratory.

Artificial samples were made by mixing HPV-negative clinicalamples (diagnosed by MY11/MY09 PCR test) with one or manyPV plasmids.

.3. PCR with specific primers

Selected reverse primers were used with MY11 in simple PCRo verify their specificity and cross-reactivity. All the HPV plasmidsere subjected separately to this PCR.

Primers were then pooled in two different multiplex reactionsnd PCR conditions were optimized using HPV DNA plasmids, arti-cial samples and HPV-positive cervical samples. Both multiplexeactions were made in a total volume of 25 �l. The reaction mix-ure contained: MgCl2 (2 mM), dNTP (200 �M), MY11 (0.2 �M), 1 Uaq DNA polymerase (Invitrogene) and specific reverse primers

0.2 �M each) in the polymerase buffer. Reverse primers for therst reaction were: HPV 16, 18, 31, 33, 35/53, 51, 52, 56, 59, 73 and2. The second reaction contained specific primers of HPV 39, 45,8 and 68.

gical Methods 196 (2014) 45– 49

Reactions were run for 40 cycles of 94 ◦C for 30 s, 59.5 ◦C (62.5 ◦Cfor the second reaction) for 20 s and 72 ◦C for 30 s. PCR cycles werepreceded by an initial denaturation at 94 ◦C for 5 min and com-pleted by a final elongation at 72 ◦C for 10 min.

2.4. Capillary electrophoresis

Capillary electrophoresis on ABI PRISM 3130 DNA analyzer wasused to identify amplified fragments. One microliter of PCR prod-uct was mixed with 0.5 �l of GS500 LIZ size standard in 12 �lof formamide and denatured before loading in capillaries filledwith POP7 polymer. The fluorescence signal was analyzed withPeakScanner v 1.0.

2.5. DNA sequencing

Clinical samples and HPV plasmids were subjected toMY11/MY09 PCR as previously described (Manos et al., 1989).The PCR products were directly sequenced using the ABI PRISM3130 DNA analyzer. For samples harboring multiple genotypes,the MY11/MY09 PCR products were cloned in “Invitrogene pCR 2.1vector” according to manufacturer’s instructions.

DNA sequencing and fragments analysis were performed in theInnovation City of Sidi Mohamed Ben Abdellah University.

2.6. Analytic sensitivity assessment

HPV plasmids with known concentration were serially dilutedand each dilution was subjected to the developed method. Thelowest concentration which elicited an interpretable signal wasdetermined for each genotype.

3. Results

Sequence alignment of the L1 region flanked by MY11 and MY09allowed the design of 15 specific primers that were fluorescentlylabeled. Every specific primer was made to generate, when usedwith MY11, a specific fragment for the corresponding type. Onereverse primer was designed to identify two genotypes: HPV35and HPV53. The theoretical sizes of generated fragments for thesegenotypes are 112 and 227 nucleotides for HPV35 and HPV53,respectively. Sequences of designed primers and the size of cor-responding fragments is reported in Table 1.

Specific reverse primers were pooled in different combinationsfor multiplex PCR and assayed on HPV DNA plasmids to assessprimers accuracy, cross-reactions and heterodimers occurrence.Finally, two separate multiplex PCR were set up for the specificidentification of 16 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52,53, 56, 58, 59, 68, 73 and 82). PCR products were subjected to cap-illary electrophoresis and fluorescence detection. Each genotypegenerates one specific signal. Low risk HPV plasmids (HPV 6 and 11)did not give any signal when tested with the developed method. Nocross-reaction was observed between HR-HPV genotypes. Experi-mentally detected fragments lengths were slightly different fromcomputational predictions for some genotypes (Table 1).

Fifty HPV-positive clinical samples were tested with the devel-oped method. From these clinical samples, 29 were found to beinfected by at least one of the sixteen targeted types. Five samplesharbored double infections when others harbored only one geno-type. The genotypes identified in clinical samples were as follow:HPV 51 (n = 10/29), HPV 53 (n = 8/29), HPV 16 (n = 5/29), HPV 58

(n = 4/29) and HPV 35 (n = 3/29). Each of HPV 31, 39, 52 and 68 werefound in one sample, whereas types 18, 33, 45, 56, 59, 73 and 82were not found in tested samples. Electrophoregrams of single andmultiple infections are presented in Figs. 1 and 2.

T. Souho, B. Bennani / Journal of Virological Methods 196 (2014) 45– 49 47

Table 1Sequences of reverse specific primers and corresponding sizes of PCR generated fragments (MY11 is used as forward primer).

Type Reverse primer sequence (5′–3′) Theoretical size (nt) Experimental size (nt)

HPV16 6-FAM-TTCTGAAGTAGATATGGCAGC 111 108HPV18 NED-ATTGCCCAGGTACAGGA 121 118HPV31 6-FAM-CACATAATCTTTAAATGGATCTTCCT 390 388HPV33 NED-TCCTTTGGAGGTACTGTTTT 359 359HPV35/53 PET-TGTCACTAGAAGACACAGCAGA 112/227 112/228HPV39 6-FAM-GCAGACTGTAGGTATCTGTAAGTGT 329 329HPV45 VIC-ACTTGTAGTAGGTGGTGGAGGG 297 300HPV51 VIC-AAACCGCAGCAGTGG 109 108HPV52 PET-CCTTTTTAACCTCAGCACAT 106 106HPV56 VIC-TTTTTCTGTTGGTGGCTG 367 363HPV58 NED-CCTTAGTTACTTCAGTGCATAATG 106 108HPV59 PET-GGGTCCTGTTTAACTGGC 377 –HPV73 VIC-AGAGCTACTAGCCTGTGTACCT 114 –HPV82 PET-GGAGTAAATGTTTGTGCAACAG 128 –HPV68 PET-AGTAGGTGCAGGGGCG 366 368

Fig. 1. Electrophoregrams representing single infections. (A) HPV 18 in artificial samples (black peak, 118 nt); (B) HPV 31 (blue peak, 388 nt), (C) HPV 52 (red peak, 106 nt)and (D) HPV 53 (red peak, 228 nt) in cervical samples. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)

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ig. 2. Electrophoregrams representing multiple infections. (A) artificial sampleith a triple HPV types; the obtained peaks a1, a2 and a3 correspond to HPV 51, 35

nd 31, respectively. (B) Clinical sample showing two peaks b1 and b2 correspondingo HPV types 51 and 53, respectively.

All MY11/MY09 PCR products were sequenced. The DNAequencing results matched perfectly with those obtained afterultiplex PCR and fragment analysis. HPV-positive samples that

ave no usable signal after fragment analysis were found afterequencing to be infected by low risk HPV genotypes (HPV 11,1 and 83). Artificial and HPV-positive clinical samples were accu-ately genotyped in all the cases (simple and multiple infections).ll targeted genotypes were detected in their respective signal, noross-reaction was observed and low risk types remained silent.

For fragment analysis, a peak is considered positive when itseight reached 300 arbitrary units. Most targeted genotypes wereetected with an interpretable signal (>300 units) at concentra-ions higher than 150 copies/�l. However, HPV 16 gave a conclusiveignal with about 50 copies/�l (Fig. 3).

. Discussion

HPV genotyping has become so important in cervical cancer pre-ention strategies, that many commercial and in-house genotyping

Fig. 3. Electrophoregrams obtained with known plasmid concentration; (A) HPV 16(45 copies); (B) HPV 18 (150 copies) and (C) HPV 35 (150 copies).

methods have been developed. PCR-based methods are the mostwidely used. Restricted fragment length polymorphism (RFLP) iseasy to perform but results are very difficult to interpret, espe-cially in case of multiple infections (Santiago et al., 2006; Zaravinoset al., 2009). Molecular hybridization with specific probes requiresmultiple-step reactions after the PCR and dedicated apparatussometimes (Kleter et al., 1999; Digene Corporation, 2004; Gheitet al., 2006; Han et al., 2006; Castle et al., 2008; Geraets et al., 2011).

The real-time PCR offers a good alternative because the reactionis performed and read out in the same apparatus but it’s limitedby the reduced number of labels applicable in one reaction; for

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xample, the Abbott real-time high risk HPV test detects 14 HRPV but identifies only 2 of them (16 and 18) (Huang et al., 2009).NA sequencing is labor intensive and not adapted in the case ofultiple infections. In sum, PCR-based methods that identify manyR-HPV are either expensive or time-consuming.

It appears necessary to develop sensitive methods applicableor high throughput screening, relatively cheap and allowing spe-ific identification of each HR-HPV genotype. For this, multiplexCR combined with capillary electrophoresis appears a good alter-ative. In this approach, PCR products are not subjected to otherhemical reactions, few amount of DNA is sufficient and the dura-ion of analysis is relatively short (Dictor and Warenholt, 2011). Inddition, fragment analysis is performed in a DNA analyzer whichan be used for many other analysis.

Specific primers were chosen on L1 gene because it’s the mostidely used for HPV types’ description. In effect, at the earliest

tages of infection, the viral DNA is present mostly in the episo-al or in both episomal and integrated forms (Arias-Pulido et al.,

006). In few cases of cervical cancer, the L1 gene is lost during DNAntegration limiting thereby the efficacy of diagnosis methods tar-eting this gene (Morris, 2005). In spite of this fact, L1 targeting istill unfailing for epidemiologic studies and also for HPV screeningn cervical cancer prevention programs.

In our knowledge, only one method of HPV genotyping is basedn multiplex PCR and capillary electrophoresis. This method tar-ets the E6/E7 region and uses 46 primers for the identification of1 genotypes (Dictor and Warenholt, 2011). In the newly devel-ped method that targets L1 gene, the number of required primerss limited to 15 specific reverse primers and a unique forwardrimer. This method was optimized using HPV plasmids before itsvaluation on clinical samples. All results show no cross-reactionsetween HPV types. The method was evaluated on clinical sam-les by studying its agreement with MY11/MY09 fragment DNAequencing. This last technique was used as standard for its effec-iveness in identifying all HPV genotypes. A total agreement wasound for all clinical samples.

Artificial samples, consisting of a mixture of HPV plasmids withPV-negative clinical samples, were tested with the new method

o assure their effective identification in real conditions. All geno-ypes were identified specifically and repeatedly in single and

ultiple infections. For samples containing high risk and low riskenotypes, only high risk types were identified and there were noross-reactions.

HPV positive clinical samples were used to evaluate the effec-iveness of the developed method in real conditions. This samplings in no case representative of the population and the genotyp-ng results do not reflect the epidemiologic profile of this region.n this HPV positive limited sampling, all HR-HPV genotypes weredentified specifically when compared to sequencing results. Lowisk genotypes did not give any signal. Curiously, HPV 51 and3 were found to be more prevalent than HPV 16 and HPV18as not found. This distribution is not in agreement with epi-emiologic data reported in Rabat (another region of Morocco) ororldwide, where HPV 16 is the most prevalent type (Bosch et al.,

008; Alhamany et al., 2010). Investigation on a larger samplings necessary to draw out more significant conclusions. This newlyeveloped method will be a helpful tool in this investigation and inhe evaluation of vaccination efficacy in our country.

. Conclusion

A new HPV genotyping method was developed. It identifies 16

R-HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58,9, 68, 73, and 82). The method is specific and reproducible. Itserformance requires low cost and simple operating procedure.hese characteristics make it a good tool for both high throughput

gical Methods 196 (2014) 45– 49

screening programs and laboratory routine use. It will providevaluable help in cervical cancer prevention programs especially inregions with low incomes.Acknowledgments

We would like to thank the staffs of gynecology-obstetrics unitand anatomo-pathology department of university Hospital HassanII Fez, for their help on specimen collection. we also thank the staffof the Innovation City of Sidi Mohamed Ben Abdellah University,Morocco for their technical support.

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