GP5+/6+ PCR followed by Reverse Line Blot Analysis Enables Rapid and High-Throughput Identification of Human Papillomavirus Genotypes

Abstract

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.High-risk human papillomavirus (HR-HPV) DNA has been shown to be present in 99.7% of cervical cancers worldwide (1,29), and the persistence of HR-HPV infection appears necessary for the development of cervical premalignant lesions and invasive cervical cancer (14,15,20,30). Therefore, HR-HPV testing may have implications for the clinical management of women with cervical lesions (25) and for primary screening for cervical cancer (17,20). In addition, several human papillomavirus (HPV)-targeted therapies have been or are being developed, and the first trials with prophylactic HPV vaccines are being conducted.To date, the detection of HPV genotypes has been done predominantly by L1 general-or consensus-primer PCR assays (3,6,12,16,24,26) and by the commercially available liquid hybridization assay Hybrid Capture 2 (HC2) (25). Generalprimer PCR assays enable the detection of a broad spectrum of mucosotropic HPV types, since the primers anneal to a highly homologous region of the HPV types spanning a polymorphic inner region, allowing specific HPV typing. Of these generalprimer PCR assays, the GP5ϩ/6ϩ and the MY09/11 PCR systems (3, 16) are the most frequently used and clinically evaluated ones. Despite the existence of more than 70 HPV genotypes, HPV testing for clinical purposes has been greatly simplified and facilitated by testing for all HR-HPV types simultaneously in one assay, i.e., HR-HPV group detection....

Human papilloma virus genotyping for the cross‐sectional and longitudinal probability of developing cervical intraepithelial neoplasia grade 2 or more

“…Genotyping was performed, blindly to histology results, by GP5+/GP6+ primed polymerase chain reaction (PCR), followed by reverse line blot 18, 19. Although GP5+/GP6+ PCR could in principle detect >40 HPV types, in this analysis, HPV genotyping was restricted to the 13 high‐risk HPV types targeted by HC2.…”

Human papillomavirus infection in women with and without cervical cancer in Nepal

“…HPV positivity was assessed by hybridization of PCR products in an enzyme immunoassay using two HPV oligoprobe cocktails which, together, detect the following 44 HPV types: HPV6, 11, 16, 18, 26, 30, 31, 32, 33, (equivalent to CP6108), and JC9710. Subsequent HPV typing was performed by reverse-line blot hybridization of PCR products, as described previously [12]. HPV types considered high-risk for this analysis included HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 [13].…”

Age-dependent prevalence of 14 high-risk HPV types in the Netherlands: implications for prophylactic vaccination and screening

“…hrHPV testing was blinded to the cytological result. hrHPV-positive samples were subjected to hrHPV typing by reverse line blot analysis of PCR products (van den Brule et al, 2002). Of screeningeligible women who participated in the POBASCAM trial (aged 29 -61 years), histological results of biopsy specimen taken at colposcopy inspection were included.…”