Development of a sensitive and specific multiplex PCR method combined with DNA microarray primer extension to detect Betapapillomavirus types

Emerging lines of evidence indicate that the cutaneous human papillomavirus (HPV) types that belong to the genus Betapapillomavirus (beta HPV) are involved in the development of nonmelanoma skin cancer. Unlike the situation for mucosal HPV types, highly sensitive and reliable methods to identify characterized cutaneous HPV types in a single assay are limited. Here, we describe a novel one-shot method for the detection of all characterized beta HPV types, namely, HPV type 5 (HPV5), 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96. This assay combines two different techniques: multiplex PCR using HPV type-specific primers for amplification of each E7 gene and array primer extension (APEX) for typing. This method has been validated using clinical samples which were analyzed simultaneously for the presence of cutaneous HPV types by two additional methods, i.e., the FAP59/64 PCR protocol and a commercially available PCR-reverse hybridization assay (PM-PCR RHA). Our data show good agreement between the results obtained with the multiplex PCR/APEX assay and the PM-PCR RHA method (overall HPV positivity of 92.2% for multiplex PCR/APEX assay versus 90.6% with the PM-PCR RHA) (kappa value, 50; 95% confidence interval, 13 to 88). In addition, the multiplex PCR/APEX assay showed higher sensitivity than the PM-PCR RHA did. This favorable feature and the high-throughput potential make this assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of cutaneous types in skin cancer.     

Development of a multiplex PCR method for detecting and typing human papillomaviruses in verrucae vulgaris

The methods for detecting and typing human papillomavirus (HPV) in most molecular epidemiological surveys of verrucae vulgaris were based on PCR followed by sequencing or hybridization. However, the amplification efficacies of different assays for the detection of HPV DNAs varied largely. In this study, a novel multiplex PCR method to detect and type the HPVs (HPV-1, -2, -27 and -57) related to verrucae vulgaris was described. This method allows detecting and typing HPV DNA simultaneously in one reaction based on the length of the PCR products after electrophoresis. The sensitivity and specificity of this multiplex PCR method was assessed with the standard template panels and the spiking sample panels, and evaluated with the clinical samples, compared with PCR assay with primer MY09/11. The results showed the novel method had reliable clinical sensitivity (97.6%) and specificity (100%), significantly higher than that of the PCR using consensus primer, MY09/11. In addition, this method can effectively detect multiple HPV infection within the lesions. This simplified, economic and time-saving multiplex PCR method provides a useful additional tool for the clinical epidemiological study of verrucae vulgaris.     

Routine genotyping of human papillomavirus samples in Denmark

In order to examine a sensitive unbiased consensus PCR with routine sequencing for HPV typing, we analysed Danish male and female patients suspected of having an HPV infection. We used the well-characterised nested PCR setting with MY09/MY11 and GP5+/GP6+ primers, followed by routine cycle sequencing. Of 1283 clinical samples from female patients based on suspected HPV infection, we found 379 (29%) negatives and 894 (70%) positives. Samples containing >5000 HPV copies/ml were genotyped by sequencing. Of the 552 HPV genotyped samples from women >15 years of age, 398 were characterised as high-risk types and the remaining 154 as low-risk types. The most commonly found high-risk types were HPV-16, HPV-31, HPV-33, HPV-18, HPV-58, and HPV-52, and the most commonly found low-risk types were HPV-6, HPV-53 and HPV-11. In addition, we observed that other typing assays could not perform as sensitively or accurately as the nested PCR/cycle sequencing method used in this study. For instance, 87 out of 552 genotyped samples could not have been typed correctly in the Hybrid Capture II assay. Of these 87 samples, 46 (53%) were considered as high-risk types.     

Clinical and Analytical Performance of the Onclarity HPV Assay Using the VALGENT Framework

As the demand for human papillomavirus (HPV)-related cervical screening increases, emerging HPV tests must be evaluated robustly using well-annotated samples, such as those generated in the Validation of HPV Genotyping Tests (VALGENT) framework. Through VALGENT, we assessed the performance of the BD Onclarity HPV assay, which detects 14 high-risk (HR) types and resolves six individual types and three groups of types. Consecutive samples from a screening population (n = 1,000), enriched with cytologically abnormal samples (n = 300), that had been tested previously with the GP5+/6+ PCR enzyme immunoassay (EIA) and the GP5+/6+ PCR LMNX assay (Diassay) were tested with the Onclarity assay. Type-specific HPV prevalences were analyzed according to age and cytological result. The accuracy of the Onclarity assay for the detection of cervical intraepithelial neoplasia grade 2+ (CIN2+) and CIN3+ was assessed relative to the GP5+/6+ EIA results by using noninferiority criteria. Overall agreement and type-specific agreement between the Onclarity assay and the GP5+/6+ LMNX assay were assessed. The prevalence of HPV types 16, 18, 31, and 45 increased with the severity of cytological results (P for trend, <0.05). For the detection of CIN2+, the Onclarity assay had a relative sensitivity of 1.02 (95% confidence interval [CI], 0.99 to 1.05; P < 0.001 for noninferiority) and a relative specificity of 0.99 (95% CI, 0.97 to 1.00; P = 0.186 for noninferiority). The kappa for agreement between the Onclarity assay and the GP5+/6+ LMNX assay for HR-HPV was 0.92 (95% CI, 0.89 to 0.94), and values for the six individual types ranged from 0.78 (95% CI, 0.68 to 0.87) for HPV-52 to 0.96 (95% CI, 0.93 to 0.99) for HPV-16. These data suggest that the Onclarity assay offers applications for clinical workstreams while providing genotyping information that may be useful for risk stratification beyond types 16 and 18.     

Simultaneous amplification and identification of 25 human papillomavirus types with Templex technology

The majority of existing human papillomavirus (HPV) genotyping assays are based on multiplex PCR using consensus or degenerate primers. We developed a Templex HPV assay that simultaneously detects and identifies 25 common HPV genotypes in a single-tube reaction using type-specific primers for the HPV-specific E6 and E7 genes. The analytical sensitivities of the Templex assay for HPV type 16 (HPV-16), -18, and -56 were 20, 100, and 20 copies per reaction mixture, respectively. The Templex assay provides semiquantitative information on each type when multiple HPV types coexist in one reaction. We tested 109 clinical cervical specimens previously evaluated with the Digene HC2 high-risk HPV DNA test and found 95.4% concordance between the assay results. The Templex assay provided type-specific results and found multiple types in 29.2% (14 of 48) of high-risk HPV-positive samples. The entire Templex procedure, including DNA extraction, can be completed within 5 hours, providing a rapid and reliable diagnostic tool for HPV detection and typing that is amenable to automation.     

A novel method of HPV genotyping using Hybrid Capture sample preparation method combined with GP5+/6+ PCR and multiplex detection on Luminex XMAP

A novel DNA detection assay comprising Hybrid Capture sample preparation, GP5+/6+ PCR with modifications and Luminex 100 detection was developed and applied to genotyping of human papillomavirus (HPV) in cervical samples. Target-specific sample preparation was performed using magnetic beads conjugated with Hybrid Capture (HC) antibody. DNA-RNA hybrids were formed between DNA target and RNA probes and captured on HC-beads. DNA on magnetic beads was amplified without elution using consensus GP5+/6+ PCR and then genotyped on Luminex beads using hybridization probes for the 17 high-risk HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, 82 and an internal control. This new sequence-specific Hybrid Capture sample preparation is fast, efficient and allows direct HPV genotyping by PCR. Compared to traditional non-sequence-specific sample preparation methods, HC sample preparation demonstrated slightly better detection of multiple HPV infections. The clinical utility of this method was demonstrated on cervical samples positive for HR HPV by the Hybrid Capture 2 (hc2) screening assay.     

Bead-based multiplex genotyping of human papillomaviruses

Typing of human papillomaviruses (HPV) by DNA hybridization procedures, such as reverse line blot (RLB) assay, is sensitive and well validated. However, the application of these assays to high-throughput analyses is limited. Here, we describe the development of multiplex human papillomavirus genotyping (MPG), a quantitative and sensitive high-throughput procedure for the identification of multiple high- and low-risk genital HPV genotypes in a single reaction. MPG is based on the amplification of HPV DNA by a general primer PCR (GP5+/6+) and the subsequent detection of the products with type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads (Luminex suspension array technology). Up to 100 different HPV types can be detected simultaneously with MPG, and the method is fast and labor saving. We detected all 22 HPV types examined with high specificity and reproducibility (the median interplate coefficient of variation was below 10%). Detection limits for the different HPV types varied between 100 and 800 pg of PCR products. We compared the performance of MPG to an established RLB assay on GP5+/6+-PCR products derived from 94 clinical samples. The evaluation showed an excellent agreement (kappa = 0.922) but also indicated a higher sensitivity of MPG. In conclusion, MPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.     

A general primer GP5+/GP6(+)-mediated PCR-enzyme immunoassay method for rapid detection of 14 high-risk and 6 low-risk human papillomavirus genotypes in cervical scrapings.

Two cocktails of digoxigenin-labeled human papillomavirus (HPV) type-specific oligonucleotide probes and an enzyme immunoassay (EIA) were used as a basis to developed a group-specific detection method for 14 high-risk (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and 6 low-risk (types 6, 11, 40, 42, 43, and 44) HPVs, following a general primer GP5+/bioGP6(+)-mediated PCR. The sensitivity of this high-risk/low-risk (HR/LR) HPV PCR-EIA ranged from 10 to 200 HPV copies, depending on the HPV type. Comparison of HR/LR HPV PCR-EIA with radioactive Southern blot hybridization using a general probe on the same PCR products derived from 417 cytomorphologically abnormal cervical scrapings resulted in an overall agreement of 96% between the two methods. Complete concordance between group-specific HR/LR detection and individual typing results for both single and multiple infections indicate the strong specificity of this HR/LR HPV PCR-EIA. Multiple infections could be predicted by comparing PCR-EIA optical density values of the cocktail probes with one of the individual oligonucleotide probes. This novel HR/LR PCR-EIA allows accurate and rapid identification of high-risk and low-risk HPV types in cervical scrapings and will facilitate HPV detection in HPV mass-screening programs.     

Detection of human papillomavirus types 6, 11, 16 and 18 in mucosal and cutaneous lesions by the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) based on the simultaneous amplification of human papillomavirus (HPV) types 6/11, 16 and 18 in a single-step procedure was developed, using primers chosen in the E6-E7 region. The specificity and sensitivity of this technique have been proved by amplifying mixtures or various amounts of plasmid-containing HPV DNA; it allowed the detection of as few as 5-25 HPV DNA copies. Application of the multiplex PCR to 71 clinical samples showed that HPV DNA was detected in 80% (45/57 cases) of mucosal biopsies and 35% (5/14 cases) of cutaneous specimens. HPV 16 was predominant in high-grade CIN whereas HPV 6 and 11 were detected more frequently in genital condylomas and laryngeal papillomas. In cutaneous Bowen's disease HPV 16, 18 or 6/11 + 16 were detected and in squamous cell carcinomas HPV 6/11 or 16 were found. After sequence amplification with primers of one HPV type, the clinical samples displayed the same HPV types but the frequency of positive and coinfected lesions increased. Thus, multiplex PCR is a valuable technique for typing HPV DNA but coinfections may be underestimated.     

Group-specific differentiation between high- and low-risk human papillomavirus genotypes by general primer-mediated PCR and two cocktails of oligonucleotide probes.

In recent years, general primer-mediated PCR assays have been developed to detect a broad spectrum of human papillomavirus (HPV) genotypes. In this study, a procedure enabling a simple group-specific differentiation of high-risk (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -54, -56, and -58) and low risk (HPV-6, -11, -34, -40, -42, -43, and 44) HPVs following an HPV general primer-mediated (GP5+/GP6+) PCR is presented. By computer-assisted sequence analysis, oligonucleotides (30-mers) specific for 19 different HPV genotypes were selected from the internal part of the 150-bp GP5+/GP6(+)-amplified region. These oligo probes were tested for specificity in a Southern blot analysis of PCR products derived from the same panel of HPV types. No cross-hybridizations were found. The sensitivities of the oligo probes varied from the femtogram level for the well-amplified HPV types like HPV-16 and -18 to the picogram level for the less-well amplified HPV types like HPV-39 and -51. These sensitivities were reached when the oligo probes were applied both individually and in a cocktail. On the basis of these results, two cocktail oligo probes that enabled a specific and sensitive differentiation between low- and high-risk HPV types were composed.     

Abundance of Multiple High-Risk Human Papillomavirus (HPV) Infections Found in Cervical Cells Analyzed by Use of an Ultrasensitive HPV Genotyping Assay

PCR methods enable the detection of a large variety of human papillomavirus (HPV) genotypes that infect the anogenital tract. However, PCR with consensus primers, general primers, and, to a lesser extent, broad-spectrum primers may underrepresent the true prevalence of HPV, especially the true prevalence of multiple infections. We compared the rate of HPV positivity determined by a broad-spectrum PCR with primers BSGP5+ and BSGP6+ (BS-PCR) coupled to an established bead-based multiplex HPV genotyping (MPG) assay with the rate of HPV positivity determined by a multiplex PCR with type-specific primers (TS-PCR) coupled to a newly developed MPG assay for 735 selected cervical scraping samples. While the primers used for the BS-PCR are located within the L1 region of the HPV genome, the primers used for the TS-PCR target the E7 gene. The overall rates of positivity for the 19 HPV types included in both assays were 60.9% and 72.2% by the BS-PCR and the TS-PCR, respectively, and the two assays found multiple infections in 34.8% and 58.0% of the specimens, respectively. Both HPV detection assays allowed the semiquantitative detection of HPV types and identified the same dominant HPV type in 66.6% of the multiple infections. In conclusion, the TS-PCR-MPG assay significantly increased the rate of detection of HPV DNA and the number of infections with multiple HPV types detected and demonstrated that the prevalence of low-copy-number HPV infections in the anogenital tract may be strongly underestimated by conventional HPV amplification methods, especially in cases of multiple infections. As a consequence, PCR-TS-MPG appears to be highly suited for analysis of the significance of multiple infections in the development of cervical cancer and for the study the natural history and the latency of HPV.Human papillomaviruses (HPV) are DNA viruses that infect cutaneous and mucosal epithelia. Until now, approximately 100 HPV genotypes have been fully characterized on the basis of the isolation of complete genomes (7), and there is evidence that a larger number exist (1). There are approximately 45 known mucosal HPV types; and these are further divided into three groups on the basis of their epidemiological association with cervical cancer: high-risk HPV (Hr-HPV) types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82), putative high-risk HPV (pHr-HPV) types (types 26, 53, and 66), and low-risk HPV (Lr-HPV) types (e.g., types 6, 11, 40, 42, 43, 44, and 70) (18). Hr-HPV types are causally associated with several malignant diseases, of which cervical cancer has particular significance, being the second most common cancer in women worldwide and the main cancer of women in most developing countries (18). Hr-HPV type DNA has been detected in 99.7% of cervical cancer tissue specimens (26), and persistent infection with an oncogenic HPV type, particularly HPV type 16 (HPV-16) or HPV-18, is recognized as a necessary cause of cervical cancer. HPV genotyping is of importance for the study of the natural history of infections with one or several HPV types and the role of HPV persistence in the progression of cervical lesions and for the monitoring of vaccine efficacy.Among HPV-positive women, 20 to 40% harbor in their cervices at least two types that were acquired simultaneously or successively (17). It remains controversial whether an infection with multiple types (referred to here as a multiple infection) is a risk factor for the persistence of HPV and for cervical lesions (20, 21). Moreover, it remains unknown whether women with, e.g., quadruple infections are at higher risk than women with double infections. Interest in multiple HPV infections has recently increased as prophylactic vaccines against HPV types 6, 11, 16, and 18 are expected to also provide partial protection against related HPV types by cross-neutralizing antibodies (12). Therefore, it is important to accurately type all HPV infections present in one patient. It will also be of particular interest to study the long-term impact of vaccination on the established equilibrium in the distribution of HPV types within immunized populations. Therefore, the sensitive, reliable, and unbiased profiling of the individual HPV types in patients with multiple infections is important to learn more about the natural history of HPV and to evaluate the effect of HPV vaccination.HPV typing based on PCR methods has continuously been improved over the past few years. One of the most common PCR uses the GP5+ and GP6+ primer pair, which targets conserved sequences within the L1 region of the virus genome flanking highly variable type-specific sequences (6). Use of PCR with this primer pair allows the amplification of a broad range of mucosal HPV types in a single reaction. The HPV genotype can be determined by analyzing the PCR product generated by sequence analysis, restriction fragment length polymorphism analysis, or hybridization with type-specific probes in different formats, such as the reverse line blot (RLB) assay (24) or the bead-based multiplex HPV genotyping (MPG) method (22). Recently, the performance of the PCR with primers GP5+ and GP6+ has been improved by addition of eight forward broad-spectrum primers and two backward broad-spectrum primers (primers BSGP5+ and BSGP6+ [BS-PCR]), which has led to a more homogeneous analytical sensitivity for the detection of genital HPV types (23). Consequently, the detection of HPV types 30, 39, 42, 44, 51, 52, 53, 68, 73, and 82, as well as the detection of multiple infections, was significantly improved by BSGP5+/BSGP6+ MPG. In addition, it has been shown that not only the use of degenerate primers (primers GP5+ and GP6+) (6) and/or consensus primers (primers MY09 and MY11) (2) but also the use of broad-spectrum primers (14) may lead to the underdetection of low copy numbers of HPV, particularly in cases of multiple infections (19).Recently, a highly sensitive, multiplex type-specific HPV E7 PCR system detecting a larger proportion of multiple infections than GP5+/GP6+ RLB has been described (9). However, the detection of PCR products was based on an array primer extension (APEX) assay, a time- and labor-intensive procedure with little high-throughput ability.In this report, we describe the development of an ultrasensitive and highly specific Luminex-based assay for the genotyping of products from the multiplex type-specific E7 PCR (TS-PCR). The results obtained by this novel high-throughput assay were compared to those obtained by the already established, sensitive BSGP5+/BSGP6+ MPG assay.     

Visual detection of high-risk human papillomavirus genotypes 16, 18, 45, 52, and 58 by loop-mediated isothermal amplification with hydroxynaphthol blue dye

A simple, rapid, sensitive, qualitative, colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB) was established to detect high-risk human papillomavirus (HPV) genotypes 16, 18, 45, 52, and 58. All initial validation studies with the control DNA proved to be type specific. The colorimetric type-specific LAMP assay could achieve a sensitivity of 10 to 100 copies at 63°C for 65 min, comparable to that of real-time PCR. In order to evaluate the reliability of HPV type-specific LAMP, the assay was further evaluated with HPV DNAs from a panel of 294 clinical specimens whose HPV status was previously determined with a novel one-step typing method with multiplex PCR. The tested panel comprised 108 HPV DNA-negative samples and 186 HPV-DNA-positive samples of 14 genotypes. The results showed that the sensitivity of HPV type-specific LAMP for HPV types 16, 18, 45, 52, and 58 was 100%, 100%, 100%, 100%, and 100%, respectively, and the specificity was 100%, 98.5%, 100%, 98.8%, and 99.2%, respectively, compared with a novel one-step typing method with multiplex PCR. No cross-reactivity with other HPV genotypes was observed. In conclusion, this qualitative and colorimetric LAMP assay has potential usefulness for the rapid screening of HPV genotype 16, 18, 45, 52, and 58 infections, especially in resource-limited hospitals or rural clinics of provincial and municipal regions in China.     

Development of a sensitive and specific nanoparticle-assisted PCR assay for detecting HPV-16 and HPV-18 DNA

Carcinoma precursor lesion caused by persistent infection of human papillomavirus (HPV) types 16 and 18 is known as a principal inducer of cervical cancer. Therefore, rapid and effective detection of HPV-16 and HPV-18 infection at early stage is an important strategy for preventing such disease. In this study, a novel duplex nanoparticle-assisted polymerase chain reaction (nanoPCR) assay was developed to detect both of the two genotypes simultaneously. Two pairs of primers for nanoPCR were designed based on the conserved region within the early 6 (E6) gene of HPV-16 and HPV-18, respectively. After optimizing reaction conditions, the nanoPCR assay displayed 10-fold more sensitive than that of conventional PCR and showed high specificity. The detection limit of nanoPCR was 1.7 × 10¹ copies/μL for HPV-16, 1.2 × 10² copies/μL for HPV-18, and no cross-reaction was detected after using other viruses or HPV subtypes as templates. Of 209 clinical samples collected from patients, as also confirmed by sequencing, the nanoPCR method gave consistent results with conventional PCR assay: 7 positives for HPV-16, 4 positives for HPV-18, and no co-infection. Here is the first report to introduce a reproducible nanoPCR assay for detecting HPV DNA with high sensitivity and specificity, which may point out a useful diagnostic tool for potential clinical application.     

Use of PGMY primers in L1 consensus PCR improves detection of human papillomavirus DNA in genital samples

The novel PGMY L1 consensus primer pair is more sensitive than the MY09 and MY11 primer mix for detection and typing with PCR of human papillomavirus (HPV) DNA in genital specimens. We assessed the diagnostic yield of PGMY primers for the detection and typing of HPV by comparing the results obtained with PGMY09/PGMY11 and MY09/MY11/HMB01 on 299 genital samples. Amplicons generated with PGMY primers were typed with the line blot assay (PGMY-line blot), while HPV amplicons obtained with the degenerate primer pool MY09/MY11/HMB01 were detected with type-specific radiolabeled probes in a dot blot assay (standard consensus PCR test). Cervicovaginal lavage samples (N = 272) and cervical scrape samples (N = 27) were tested in parallel with both PCR tests. The PGMY-line blot test detected the presence of HPV DNA more frequently than the standard consensus PCR assay. The concordance for HPV typing between the two assays was 84.3% (214 of 255 samples), for a good kappa value of 0.69. Of the 177 samples containing HPV DNA by at least one method, 40 samples contained at least one HPV type detected only with PGMY-line blot, whereas positivity exclusively with the standard consensus PCR test was found for only 7 samples (P < 0.001). HPV types 45 and 52 were especially more frequently detected with PGMY than MY primers. However, most HPV types were better amplified with PGMY primers, including HPV-16. Samples with discordant results between the two PCR assays more frequently contained multiple HPV types. Studies using PGMY instead of MY primers have the potential to report higher detection rates of HPV infection not only for newer HPV types but also for well-known genital types.     

Detection and typing of human papillomavirus by e6 nested multiplex PCR

A nested multiplex PCR (NMPCR) assay that combines degenerate E6/E7 consensus primers and type-specific primers was evaluated for the detection and typing of human papillomavirus (HPV) genotypes 6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66, and 68 using HPV DNA-containing plasmids and cervical scrapes (n = 1,525). The performance of the NMPCR assay relative to that of conventional PCR with MY09-MY11 and GP5+-GP6+ primers, and nested PCR with these two primer sets (MY/GP) was evaluated in 495 cervical scrapes with corresponding histologic and cytologic findings. HPV prevalence rates determined with the NMPCR assay were 34.7% (102 of 294) in the absence of cervical intraepithelial neoplasia (CIN 0), 94.2% (113 of 120) in the presence of mild or moderate dysplasia (CIN I/II), and 97.8% (44 of 45) in the presence of severe dysplasia (CIN III). The combination of all four HPV detection methods applied in the study was taken as "gold standard": in all three morphological subgroups the NMPCR assay had significantly (P < 0.0001) higher sensitivities than the MY09-MY11 and GP5+-GP6+ assays and sensitivities comparable or equal to those of the MY/GP assay. All 18 HPV genotypes investigated were detected among the clinical samples. The ratio of high- to low-risk HPV genotypes increased from 4:1 (80 of 103) in CIN 0 to 19:1 (149 of 157) in CIN I to III. Multiple infections were detected in 47.9% (124 of 259) of the patients. In conclusion, the novel NMPCR method is a sensitive and useful tool for HPV DNA detection, especially when exact HPV genotyping and the identification of multiple HPV infections are required.     

Clinical validation of Anyplex™ II HPV HR Detection according to the guidelines for HPV test requirements for cervical cancer screening

BackgroundAnyplex™ II HPV HR Detection (Seegene, Seoul, Korea) is a multiplex real-time PCR using tagging oligonucleotide cleavage and extension (TOCE) technology for simultaneous detection and genotyping of 14 high-risk (HR) HPV types, including HPV16 and HPV18.ObjectivesTo evaluate whether the clinical performance and reproducibility of Anyplex™ II HPV HR Detection meet the international consensus guidelines for HPV test requirements for cervical cancer screening [1].Study designThe clinical performance of Anyplex™ II HPV HR Detection for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was determined relative to that of the reference assay, i.e., HR HPV GP5+/6+-PCR-EIA, by analysis of a total of 879 cervical liquid based cytology (LBC) specimens from a screening population, of which 60 were from women with CIN2+. The intra-laboratory reproducibility and inter-laboratory agreement were determined on 509 LBC samples, of which 172 were positive by the reference assay.ResultsAnyplex™ II HPV HR Detection showed a clinical sensitivity for CIN2+ of 98.3% (59/60; 95% CI: 89.1–99.8) and a clinical specificity for CIN2+ of 93.6% (764/816; 95% CI: 89.8–96.1). The clinical sensitivity and specificity were non-inferior to those of HR HPV GP5+/6+-PCR-EIA (non-inferiority score test: P = 0.005 and P = 0.023, respectively). Both intra-laboratory reproducibility (96.8%; 95% CI: 95.3–98.1; kappa value of 0.93) and inter-laboratory agreement (96.0%; 95% CI: 94.3–97.4; kappa value of 0.91) were high.ConclusionsAnyplex™ II HPV HR Detection performs clinically non-inferior to HR HPV GP5+/6+-PCR-EIA. Anyplex™ II HPV HR Detection complies with international consensus validation metrics for HPV DNA tests for cervical cancer screening [1].     

GeXP多重PCR技术用于人乳头瘤病毒HPV检测和分型的研究

目的建立一种基于GenomeLab^TM GeXP（GenomeLab eXpress Profiling）遗传分析系统的人乳头瘤病毒（HPV）分型新技术。方法针对5种中国人常见的HPV亚型（HPV6,11,31,33和52）的基因序列进行比对分析,设计型特异性引物,建立并优化GeXP多重PCR反应体系,评价其特异性和灵敏性,并应用于30份临床标本检测。结果本研究建立的GeXP多重PCR技术能够成功检测和鉴别5种不同亚型的HPV病毒,灵敏度高,特异性好。结论成功建立了一种新的快速同时检测和鉴别5种HPV亚型病毒的多重PCR方法,适用于临床HPV感染的实验室诊断与流行病学研究。     

A non-radioactive PCR enzyme-immunoassay enables a rapid identification of HPV 16 and 18 in cervical scrapes after GP5+/6+ PCR

In previous studies, general primer mediated PCR (GP5+/6+ PCR) was applied successfully to detect a broad spectrum of human papillomaviruses (HPV) in cervical scrapes. In order to facilitate PCR based HPV detection and typing, a colourimetric microtitre plate based hybridisation assay was developed. The method utilised one biotinylated primer (bio-GP6+) in the GP-PCR. Biotinylated PCR products were captured on streptavidin coated microtitre plates, denaturated and hybridised to digoxigenin (DIG) labelled HPV specific internal oligo probes. The DIG labelled hybrids were detected using an enzyme immunoassay (EIA). Since HPV 16 and 18 are the most common HPV types found in cervical carcinomas, this approach was initiated for these two types. Cross-hybridisation reactions were not detected when the specificity of this PCR-EIA for HPV 16 and 18 was tested on a panel of 20 different HPV genotypes. The sensitivity of the assay was found to be between 10 and 100 HPV 16 and 18 viral genomes in a background of 100 ng cellular DNA. This was similar to the detection limit of Southern blot analysis of PCR products with radioactively labelled oligonucleotides. A group of cytomorphologically normal (n = 89) and abnormal (n = 96) cervical scrapes were composed of HPV 16 and HPV 18 positive and HPV negative scrapes. All HPV 16 and 18 positive smears were detected by PCR-EIA. These results indicate that PCR-EIA has the potential for a rapid and sensitive HPV DNA test for day-to-day routine examination of cervical scrapes. © 1996 Wiley-Liss, Inc.     

GP5+/6+ PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes

In this study, we developed a simple and fast typing procedure for 37 mucosotropic human papillomavirus (HPV) types using a nonradioactive reverse line blotting (RLB) procedure for general primer (GP5+/6+) PCR products. This system has the advantages not only that in a simple format, up to 42 PCR products can be simultaneously typed per membrane per day, but also that after stripping, the membranes can be easily rehybridized at least 15 times without a loss of signal. RLB appeared highly specific, and its sensitivity was identical to that of conventional typing performed with type-specific oligonucleotide probes in an enzyme immunoassay (EIA). The performance of RLB typing was evaluated with samples of HPV-positive cervical scrapings (n = 196) and biopsies of cervical premalignant lesions (n = 100). The distribution of HPV genotypes detected in these samples was in line with the distribution expected on the basis of literature data. In addition, RLB and EIA typing procedures were compared for the typing of high-risk HPV types in GP5+/6+ PCR products of 210 cervical scrapings from high-risk HPV-positive women who participated in a population-based screening program. The typing procedures had an excellent overall agreement rate of 96.5% (kappa value, 0.77). RLB was successful in detecting multiple HPV infections as well as single infections. In conclusion, the GP5+/6+ PCR-RLB procedure appeared to be a reliable and simple approach that may be of great value for large epidemiological studies, population-based cervical cancer screening programs, and vaccination trials that require high-throughput HPV typing.