6+ primer sets improves detection of HPV DNA in cervical samples

Journal of Virological Methods 122 (2004) 87–93

Nested PCR with the PGMY09/11 and GP5+/6+ primer sets improves detection of HPV DNA in cervical samples Andrea L. Fuessel Hawsa , Qin Hea , Peter L. Radyc , Lifang Zhangb , James Gradyb , Thomas K. Hughesa , Kendra Stissera , Rolf Koniga , Stephen K. Tyringa,c,∗ a

Departments of Microbiology/Immunology, University of Texas Medical Branch, Galveston, TX 77030, USA b The Office of Biostatistics, University of Texas Medical Branch, Galveston, TX 77030, USA c Department of Dermatology, University of Texas Medical School at Houston, Houston, TX 77030, USA Received 31 March 2004; received in revised form 4 August 2004; accepted 5 August 2004

Abstract Based on epidemiological and research evidence, HPV has a causal role in cervical carcinogenesis. Several HPV detection methods exist to date; the most commonly used method for detection of genital HPVs consists of nested PCR using the MY09/11 and GP5+ /6+ primer sets (MY/GP+ ). Recently, the PGMY09/11 primer set, a modified version of the MY09/11 primer set, was introduced for single PCR and was found to detect a wider range of HPV types. The next logical step was taken and the efficacy of nested PCR using the PGMY09/11 and GP5+ /6+ primer sets (PGMY/GP+ ) to detect HPV in cervical samples was evaluated. In this comparative study, nested PCR using the novel PGMY/GP+ primer set combination was found to be more type sensitive than the nested PCR with the MY/GP+ primer sets, detecting a wider range of HPV types, low copy HPVs, and better characterizing samples infected with multiple strains of HPV. Standardization and use of the PGMY/GP+ PCR system could aid physicians in providing more efficient HPV screening and better treatment for patients. © 2004 Elsevier B.V. All rights reserved. Keywords: Human papillomavirus; Detection; PCR; PGMY; GP+

1. Introduction Human papillomavirus (HPV) infection remains a prominent concern in both the research and medical fields. It is capable of infecting the basal epithelial cells of all men and women worldwide, regardless of age or race, and can cause a wide variety of clinical manifestations, which can be either benign or malignant (Burd, 2003). Although HPV has been associated with many diseases, cervical cancer has particular significance, being the second most common cancer in women worldwide, the third most common cancer in women in the United States, and the principal cancer of women in most developing countries (Burd, 2003; Munoz et al., 2003). ∗

Corresponding author. Tel.: +1 713 528 8818; fax: +1 281 335 4605. E-mail address: [email protected] (S.K. Tyring).

0166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2004.08.007

More than 230 HPV types have been recognized to date on the basis of DNA sequence data showing genomic differences; 85 HPV genotypes are fully characterized (Burd, 2003; de Villiers, 1999). There are approximately 40 known genital HPVs, which are further divided into high-risk (HR-HPV; oncogenic: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82; probably carcinogenic: 26, 53, 66) and low risk (LR-HPV; e.g. 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and candHPV89/CP6108) groups for cervical cancer (Terai and Burk, 2002; Munoz et al., 2003). HPV type 16 is the most common HPV type detected in cervical cancers worldwide, followed by HPV 18 (Munoz, 2000). Several PCR based HPV detection methods exist to date; however the most commonly used PCR methods for the detection of genital HPVs have used the MY09/11 and GP5+ /6+ primer sets (MY/GP+ ) (Strauss et al., 2000;

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Evander et al., 1992; de Roda Husman et al., 1995; Manos et al., 1989). These primers target the conserved L1 region of the virus genome, thus allowing the detection of a broad range of HPV types. This method also enables the type of HPV to be determined by sequencing of the generated PCR product (Rady et al., 1993, 1995). The advent of this method from the previous single PCR approach enabled a wider range of HPV type detection, especially in samples that were multiply infected (Evander et al., 1992). Since the design of the MY09/11 primers, many more mucosal and genital HPV types have been discovered. Gravitt et al. (2000) took the next logical step and improved the MY09/11 primer set by taking into account the consensus L1 sequence for the (then known) approximately 20 HPV types. The PGMY09/11 primers each consist of oligonucleotide pools which bind in the same region the MY09/11 primer set binds; however, the fact that they are not degenerate primer sets provides a measure of quality assurance in primer design. Additionally, the design of the primer pools reduced the significant internal secondary structure formation of the oligonucleotides, and reduced internal priming. Furthermore, the PGMY 09/11 primer set has exhibited higher sensitivity and a broader range of HPV type detection, especially with regard to multiply infected samples (Gravitt et al., 2000). The PGMY09/11 primer set detected HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 at least 25% more often than the MY09/11 primer set (Gravitt et al., 2000). Nested PCR with the PGMY09/11 and GP5+ /6+ primer sets has not yet been evaluated nor applied (PGMY/GP+ ). In this study, the next logical step was taken; the HPV diagnostic value of the novel PGMY/GP+ primer combination in a nested PCR system was established, evaluated, and applied. Specifically, nested PCR with the PGMY/GP+ primer sets was compared to the established nested PCR standard using the MY/GP+ primer sets. The PGMY/GP+ nested PCR system was found to be statistically more type-sensitive than the MY/GP+ nested PCR system, being able to detect a wider range of HPV types, especially when characterizing multiply infected samples. The PGMY/GP+ nested PCR system was then applied in evaluating HPV infection in cervical samples obtained from a high-risk population.

2. Materials and methods 2.1. Study population and sample collecting Women seeking treatment at the University of Texas Medical Branch in Galveston (UTMB) Gynecologic Oncology clinic, and who had a history of cervical dysplasia, were asked to participate in this study. Informed consent was obtained from the patients by trained interviewers. A Papanicolau (Pap) smear was obtained from each of the women using Cervical Sampler (Digene, Gaithersburg, MD). The study protocol was approved by the Institutional Review Board at UTMB.

Pap smear slides were evaluated by the Cytopathology lab at UTMB. Cellular changes were classified by the pathologists according to the Bethesda classification system as negative (normal), atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), or suggestive of cancer. 2.2. DNA extraction and sample DNA quality assessment DNA extraction was conducted in a designated “DNA extraction” hood using the PuregeneTM DNA Isolation Kit for Buccal Cells by Gentra Systems. The manufacturer’s protocol was followed with the following modifications: 3 ␮L Proteinase K Solution (20 mg/mL) was used during cell lysis, 2 ␮L of Poly Acryl carrier (Molecular Research Center, Inc) was used during DNA precipitation instead of the Gentra Glycogen Solution, and DNA was hydrated in 100 ␮L of DNAse/RNAse free water instead of the DNA hydration solution. All extracted samples were stored at 4 ◦ C until further use. Amplifiable quality of DNA was confirmed by using 5 ␮L of each DNA sample for PCR assay targeting the human beta-globin gene with the PC04 and GH20 primers adapted from Gravitt et al. (2000). The PCR products were visualized by 2% agarose gel electrophoresis. 2.3. HPV detection and typing The samples were screened for the presence of HPV using the standard nested PCR approach consisting of the MY09/11 primer set (primary PCR) described by Manos et al. and the GP5+ /6+ primer set (secondary PCR) described by de Roda Husman et al. (Manos et al., 1989; de Roda Husman et al., 1995; Gravitt et al., 2000; Strauss et al., 2000; van den Brule et al., 2002). Briefly, 5 ␮L of each sample was amplified with MY09/11 primers (5 pmol each) and 2 U of AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) using the Molecular BioProducts EasyStartTM PCR tubes (1× PCR buffer II, 2 mM MgCl2 , 200 ␮M [each] dATP, dCTP, dGTP, and dTTP). Additional MgCl2 was added to each reaction for a final concentration of 6 mM (Gravitt et al., 2000). Amplifications were performed in a Biometra TRIO-Thermoblock TB-1 thermocycler using the following specifications: 95 ◦ C for 2 min, 40 cycles of 95 ◦ C for 1 min, 55 ◦ C for 1 min, and 72 ◦ C for 1 min. This was followed by a final extension of 10 min at 72 ◦ C, and then storage at 4 ◦ C. The secondary PCR consisted of amplification of 5 ␮L of the primary PCR product using the GP5+ /6+ primers (5 pmol each). PCR buffers and reagents were identical to the primary PCR, with the exception of MgCl2 having a final concentration of 3.5 mM. Amplifications were performed in the same machine as described above using the following specifications: 94 ◦ C for 2 min, and 40 cycles of 94 ◦ C for 45 s, 48 ◦ C for 4 s, 38 ◦ C for 30 s, 42 ◦ C for 5 s, 66 ◦ C for 5 s, and 71 ◦ C for 1.5 min. This was followed by a final extension of 10 min at 72 ◦ C, and then storage at 4 ◦ C (van den Brule et al., 2002).

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Similarly, the samples were screened for the presence of HPV using a nested PCR approach consisting of the PGMY 09/11 primer set (primary PCR) described by PE Gravitt et al. and the GP5+ /6+ primer set (secondary PCR) described by de Roda Husman et al. (de Roda Husman et al., 1995; Gravitt et al., 2000). Briefly, 5 ␮L of sample DNA was amplified with PGMY09/11 primers (10 pmol each). All other PCR buffers, reagents, and amplification profiles were identical to the MY09/11 primary PCR described above, with the exception of the MgCl2 final concentration being 4 mM in each reaction (Gravitt et al., 2000). The secondary PCR consisted of amplifying 5 ␮L of the PGMY09/11 PCR product with the GP5+ /6+ primers as described above. The PCR products were electrophoresed using a 2% low-melting point gel (NuSieve GTG Agarose) in 1× TBE buffer, stained with ethidium bromide and photographed under UV-transillumination. The bands from any samples that were positive for the primary and/or secondary PCR were isolated and purified using the Bio-Rad Quantum PrepTM Freeze ’N Squeeze DNA Gel Extraction Spin Columns and the Amicon® Microcon® -PCR Centrifugal Filter Devices by Millipore. The purified PCR products were then submitted for automated DNA sequencing (Sealy Center for Molecular Science Recombinant DNA Laboratory, UTMB, Galveston, TX). The sequences were compared with HPV genomes deposited in the NCBI-GenBank using the BLAST program (NCBI). As a measure for quality control, samples were randomly chosen for repeat analysis. Results of the repeat analysis were concurrent with the initial analysis. 2.4. Cloning Samples for which a clean sequence could not be obtained were subjected to cloning by following the manufacturer’s protocol for the InvitrogenTM TOPO TA Cloning® Kit for Sequencing. Six clones for each sample were selected for plasmid purification followed by automated DNA sequencing. 2.5. Sensitivity The sensitivity of the PGMY09/11 and GP5+ /6+ nested PCR was compared to that of the MY09/11 and GP5+ /6+ . The concentration of a puc19 plasmid containing the complete genome of HPV 6 (kindly provided by Dr. E.M. De Villiers, Heidelberg, Germany) was determined precisely using the Molecular Probes PicoGreen® dsDNA Quantitation Kit. Nested PCR using the MY09/11 and GP5+ /6+ primer sets and the PGMY09/11 and GP5+ /6+ primer sets was conducted, as described above, on samples containing 500 ng Human Placental DNA (Sigma-Aldrich) along with 1, 5, 10, 100, 1000, 10,000, and 100,000 copies of the HPV 6 containing plasmid. This assay was done in triplicate.

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2.6. Statistical analysis Paired T-tests were used to compare the number of HPV types identified between the PGMY/GP+ and the MY/GP+ systems. Kappa statistics were calculated to measure the agreement of the primer systems beyond that expected by chance. For most purposes, values of kappa greater than 0.75 may be taken to represent excellent agreement beyond chance, and values between 0.40 and 0.75 represent fair to good agreement. (Fleiss, 1981). McNemar’s Chi-square tests for matched pair data were performed to test for unequal distribution of discordant results when comparing high-risk HPV types, low-risk HPV types, and non-classified HPV types that were detected by the various PCR primer combinations.

3. Results 3.1. HPV typing using nested and single PCR A total of 37 cervical smear samples obtained from women living in Galveston, who have cervical dysplasia or who have a history of cervical dysplasia, were analyzed. They were first analyzed by nested PCR using the MY09/11 primers (primary PCR), followed by the GP5+ /6+ primers (secondary PCR). After electrophoresis, 26 samples contained a band of near identical size as the expected HPV PCR product; however, sequencing and NCBI Blast results of the PCR product revealed that only 11 samples were HPV positive, detecting 12 HPV types, and 15 samples matched only with human genomic sequences (Human BAC clone RP11-420A23 from 4, NCBI GenBank accession number AC108062, was detected most frequently). Twenty-six samples were positive for the secondary PCR (GP5+ /6+ ), detecting fifteen HPV types, for a total of 17 different HPV types being detected. Eight samples contained multiple infections. After cloning, a maximum of two HPV types were detected in a single sample (Table 1, Fig. 1). The same samples were analyzed by nested PCR using the PGMY09/11 primers (primary PCR), followed by the GP5+ /6+ primers (secondary PCR). Eleven samples were positive during the primary PCR, detecting twelve HPV types; no false-positives were obtained (Table 1). The PGMY PCR detected HPV type 45 while the MY PCR did not; conversely, the MY PCR detected HPV type 54 while PGMY PCR did not. Twenty-eight samples were positive for the secondary PCR, detecting an additional two samples infected with HPV and 20 HPV types, for a total of 23 HPV types being detected by the system. The additional HPV types detected by the PGMY system included HPV 11, 31, 54-AE9, 67, 89, and SW1. Twelve samples were multiply infected, with a maximum of three HPV types detected in each sample. It should be noted, however, that while often a same HPV type was detected with both systems in a multiply infected sample, the companion HPV types differed, with the PGMY

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Table 1 HPV detection and typing results using nested PCR comparing MY/GP+ and PGMY/GP+ nested PCR Sample

PGMY

PGMY/GP+

MY

MY/GP+

Current pathologya

Past pathologyb

G19-02 G20-02 G21-02 G22-02 G23-02 G25-02 G26-02 G27-02 G28-02 G29-02 G30-02 G32-02 G33-02 G34-02 G36-02 G38-02 G39-02 G40-02 G41-02 G42-02 G43-02 G44-02 G45-02 G46-02 G47-02 G48-02 G49-02 G50-02 G51-02 G52-02 G53-02 G54-02 G55-02 G56-02 G57-02 G58-02 G59-02

Neg 52 Neg Neg Neg Neg 16 Neg Neg 45 Neg Neg Neg Neg 33 Neg Neg Neg Neg Neg Neg 39 Neg Neg Neg Neg 70, 71, 85 Neg 18 35 Neg Neg 61 Neg Neg 16 72

16, 62 52 18, 66 16 33 18 16 18, 62, 66 16, 54, 54-AE9 45 16, 31 16, 58 16, 58, 89c 11, 16, 18 33 16, 18, 62 85 Neg 6 54 Neg 39 Neg Neg Neg Neg 70, 85 Neg 18 35 Neg Neg 61, 72 66 67, SW1 16 72

Neg 52 Neg Neg Neg Neg Neg Neg 54 Neg Neg Neg Neg Neg 33 Neg 85 Neg Neg Neg Neg 39 Neg Neg Neg Neg 70, 71, 85 Neg 18 35 Neg Neg 61 Neg Neg 16 72

16, 62 52 Neg Neg 33 18 16 16, 18 16, 54 45 33 58 58, 70 16 33 16, 18 85 Neg 6 18, 54 Neg 39 Neg Neg Neg Neg 85 Neg 18 35 Neg Neg 72 66 52 16 72

Neg CIN 1 CIN 3 CIN 2/3 CIN 3 CIN 3 CIN 1 Neg CIN 1 Neg CIN 1 Neg Neg Neg CIN 3 CIN 1 CIN 2 Neg CIN 3 Neg Neg ASCUS Neg Neg Neg Neg Neg Neg ASCUS Neg Neg CIN 1 CIN 3 CIN 1 Neg CIN1 Neg

CIN 3 n/a n/a n/a n/a n/a CIN 3 CIN 3 CIN 3 n/a CIN 3 CIN 3 CIN 3 CIN 3 n/a n/a CIN 3 CIN 1 n/a CIN 1 CIN 3 CIN CIN 1, CIN 3 CIN 3 CIN 1 ASCUS CIN 1 ASCUS CIN 3 CIN 1 ASCUS, CIN 1 n/a n/a n/a AGUS n/a CIN 2–3

a

Pathology results obtained at time of sample collecting for the study. Patient cervical history; women generally obtained treatment at some point in time after the “past pathology” was diagnosed but before the “current pathology” was evaluated. CIN, cervical intraepithelial neoplasia; Neg, within normal limits; ASCUS, atypical squamous cells of undetermined significance; AGUS, atypical glandular cells of undetermined significance. c HPV 89 is a candidate type whose L1 sequence matches isolate CP6108 (Terai and Burk, 2002). b

system usually detecting additional HPV genotypes in the sample. When comparing the nested PCR systems in terms of detecting the number of HPV types per sample, no statistical difference was found for the first step of PCR (MY versus PGMY); agreement between the first PCR steps was strong (data not shown; percent agreement = 94.6%; kappa = 0.83; 95% CI = 0.59, 1.00). Although not statistically significant, there were two samples with discordant results, both of which were positive with the PGMY09/11 primer set only. When comparing the nested PCR systems to each other as a whole (MY/GP+ versus PGMY/GP+ ), the PGMY/GP+ PCR system was found to detect on average 0.30 (SD = 0.62; P = 0.006) more HPV types per sample than the MY/GP+ system. The agreement between the systems remained strong (data not shown; percent agreement = 94.6%; kappa = 0.86;

95% CI = 0.68, 1.00). The two previously discordant results remained discordant, with the same findings described for the first step of PCR above. When comparing the systems in terms of detecting HPV risk-types in each of the samples, each sample was coded for the HPV risk types it contained as follows: HR-HPV if it contained at least one of the following HPV types: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, or 82; LR-HPV type if it contained at least one of the following HPV types: 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, or candHPV89/CP6108; undetermined risk HPV type (URHPV) if it contained at least one of the following HPV types: 34, 57, 62, 67, 71, 83, 85, or isolate sw1. For example, if a sampled contained both HPV 16 and 6, it was coded as both HR-HPV and LR-HPV. The two nested PCR systems exhibited an 89.2% agreement in detecting HR-HPV types (kappa

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Fig. 1. Comparison of HPV types detected by nested PCR using the MY/GP+ and PGMY/GP+ primer sets. Comparison of HPV types detected by nested PCR using the MY/GP+ or PGMY/GP+ , primer sets. Grey bar, detected with MY/GP+ , black bar, detected with PGMY/GP+ . These type-specific results include positive results detected in samples infected with both single and multiple HPV types.

= 0.78; 95% CI = 0.58, 0.98) with the PGMY/GP+ system classifying two samples as being infected HR-HPV while the MY/GP+ system classified them as HPV negative, and the MY/GP+ system also classified two samples as being infected with HR-HPV while the PGMY/GP+ system classified them as HPV negative. When comparing their ability to detect a LR-HPV type, the two systems exhibited a 97.3% agreement (kappa = 0.92; 95% CI = 0.76, 1.00), with the PGMY/GP+ system classifying one sample as being infected with a LRHPV type while the MY/GP+ system classified this sample as HPV negative. When comparing their ability to detect URHPVs, the two systems exhibited a 91.9% agreement (kappa = 0.63; 95% CI = 0.23, 1.00), with the PGMY/GP+ system classifying three samples as being infected with an UR-HPV while the MY/GP+ system classified these samples as HPV negative. When comparing the systems in terms of detecting LR-HPV and UR-HPV types together, the two systems exhibited an 89.2% agreement (kappa = 0.74; 95% CI = 0.52, 0.97), with the PGMY/GP+ system classifying four samples as being infected with either a LR-HPV or an UR-HPV while the MY/GP+ system classified these samples as being HPV negative; additionally, the PGMY/GP+ system detected on average 0.16 (or approximately four types per 25 patients, P = 0.03) more low-risk or undetermined-risk HPV types per sample than the MY/GP+ system (data not shown). The two systems were then compared by classifying hierarchically samples in terms of cancer risk group as follows: HR-HPV, UR-HPV (without concomitant coinfection with a high-risk type), LR-HPV (without concomitant coinfection with a high-risk or undetermined-risk type), or HPV negative. The two systems exhibited an 89.2% agreement (kappa = 0.82; 95% CI = 0.65, 0.99). With the PGMY/GP+ system classifying two samples as HR-HPV while the MY/GP+ system classified these two samples as HPV negative; conversely, the MY/GP+ system classified two samples as being infected with HR-HPVs while the PGMY/GP+ system clas-

sified one of these samples as being infected with a LR-HPV and one sample as being infected with an UR-HPV. 3.2. Sensitivity assay results The sensitivity of both the MY/GP+ and the PGMY/GP+ systems was determined. In the presence of 500 ng of human placental DNA, 1, 5, 10, 100, 1000, 10,000, and 100,000 copies of a plasmid containing the entire HPV 6 genome were amplified using both the MY/GP+ and the PGMY/GP+ systems. The complete analysis was performed in triplicate. The primary PCR of the MY/GP+ system detected HPV DNA at 10,000 copies (data not shown). However, gel electrophoresis of PCR amplicons from 1, 5, 10, 100, and 1000 copies gave the impression that these samples were HPV positive. Upon sequencing the purified amplicons, only human genomic DNA was identified (Homo sapiens BAC clone RP11420A23 from 4). The primary PCR of the PGMY/GP+ system unambiguously showed detection of HPV DNA on the gel at 10,000 copies, which was confirmed by sequencing. Two times out of three, the secondary PCR of the MY/GP+ system detected HPV DNA at five copies, and once it detected HPV DNA at one copy. The secondary PCR of the PGMY/GP+ system showed consistently detection of HPV DNA at one copy.

4. Discussion In this study, the PGMY/GP+ nested PCR system for genital HPV detection from cervical samples was established. The PGMY/GP+ system was compared to the previously established nested PCR using the MY/GP+ primer combination, and the PGMY/GP+ system was found to statistically detect more HPV types. On average, the PGMY/GP+ system detected an additional HPV type for every three samples an-

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alyzed. Additionally, it is capable of detecting even one copy of HPV in a PCR reaction. The PGMY/GP+ system does not non-specifically amplify human DNA, thus preserving time and cost. In this study, it has been shown that the combination of the PGMY primer set with the GP5+ /6+ primer set further enhances the ability to characterize HPV infected samples. It is interesting that neither the PGMY/GP+ nor the MY/GP+ nested PCR system detected HPV type 43 in sample G42, whereas the GP+ single PCR did. In this case, many factors influencing the microenvironment of the PCR reaction become apparent. In the single GP+ PCR, the original DNA sample was diluted 1:20 in the PCR reaction; here, it is very possible that the HPV type 43 was present sufficiently to be “seen” by the GP+ primers, and was amplified preferentially. With the nested PCR systems, however, the first PCR reaction (the MY and PGMY primers) could have preferentially amplified HPV types 18 and 54; in the second step of the nested PCR reaction, a dilution factor of 1:400 must be considered. At this point, either the HPV 43 was not present in the second PCR reaction or the concentrations of HPV types 18 and 54 (amplified by the first round of PCR) masked the HPV 43, and it was not detected. There is a clear need for an improved HPV detection and typing system in order to reduce the potential of “false negative” results due to missing an infecting HPV type, to reduce “false positive” results due to non-specific amplification of genomic DNA, to increase the span of the HPV types detected, to better characterize multiply infected samples, and to conserve time and cost. New HPV types are continuously being discovered, and it is likely that the known number of HPVs is much smaller than the actual number of HPVs in circulation. An ideal HPV detection and typing system would be specific for HPV, sensitive and reliable enough to consistently detect HPV in cervical samples, have the ability to thoroughly characterize multiply infected samples, and yet be flexible enough to detect novel HPV types. We have found that in the presence of low viral loads, the MY09/11 primer set will non-specifically amplify human DNA; this PCR product size is often the same as the expected HPV product size. This can interfere with HPV typing by giving a false HPV positive MY09/11 PCR product when analyzing PCR results by gel electrophoresis. Sequencing of this “false positive” product reveals the product to contain human sequences, most frequently the BAC clone RP11420A23 from 4. Additionally, this method does not give a true representation of the HPV types in a sample, as it often misses an HPV type in a multiply infected sample, giving the impression that the sample is singly infected. The potential to miss a high-risk HPV type is especially undesirable. Hybridization-based methods, such as the commercially available hybrid capture (HC2 and HC3) assay that is widely used in clinics to detect HPV in cervical scrapings, also have large limitations. Although they are sensitive, hybridizationbased methods are limited in their detection capabilities by the hybridization probe mixtures they utilize, and do not allow HPV typing. Furthermore, studies have shown that CIN2/3

can be found in 11–20% of women testing negative for highrisk HPV by HC2 (Pisal et al., 2003; Lonky et al., 2003; Guyot et al., 2004). PCR showed that the negative HC2 results were not due to novel or other HPV types not tested by HC2, but were concluded to be the result of sampling error (Lonky et al., 2003; Guyot et al., 2004). When treating patients with CIN, it is important to monitor HPV persistency, as it is a reliable indicator of CIN or cancer recurrence risk for the patient; however, only typespecific analysis, which is achievable with PCR-based HPV diagnosis techniques, can differentiate between persistence and a new HPV infection (Nobbenhuis et al., 2001; Speich et al., 2001). Furthermore, it has been shown that PCR systems using multiple primers, such as PGMY09/11, are more robust in detecting multiple infections than systems using single consensus primers; this is especially true in case when one HPV type is present in large amounts (Iftner and Villa, 2003). In conclusion, the PGMY/GP+ PCR system has been shown to be more type-specific, detecting more HPV types per sample, and thus better characterizing multiply infected samples. The high sensitivity of the PGMY/GP+ system allows detection of low copy HPV and a wider HPV type sensitivity, especially in determining the viral types in multiply infected samples. Standardization and use of the PGMY/GP+ PCR system could aid physicians in providing better treatment and more efficient screening for patients. Acknowledgements These studies were supported by the NCI, grant #1RO3 CA030730 (SKT) and in part by the NIH Predoctoral Training Program in Mucosal Immunology T32-AI07626 01A1 (RK).

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