Human papillomavirus (HPV) DNA detection and typing are important for diagnosis and management of HPV-associated diseases. One of the most commonly used PCR methods, GP5؉/6؉, shows weaknesses in amplifying certain types. To circumvent this limitation, we developed and validated broad-spectrum primers targeting the GP5؉/6؉ region. The addition of eight upstream and two downstream BSGP5؉/6؉ (BS) primers improved amplification of plasmids of 14 genital HPV types 10-to 1,000-fold versus GP5؉/6؉ PCR without altering sensitivity for the 10 others. For these 24 types, an analytic sensitivity of <1,000 plasmid copies in the presence of 100 ng cellular DNA was obtained. Additionally, we integrated an internal ␤-globin PCR into both HPV PCR systems, allowing simultaneous DNA quality control without affecting the sensitivity of HPV detection. Furthermore, we describe five additional low-risk HPV probes used in multiplex HPV genotyping (MPG) for simultaneous identification of all 15 high-risk, 3 putative high-risk, and 9 low-risk HPV genotypes. The performance of BSGP5؉/6؉ multiplexed with ␤-globin primers was compared to that of standard GP5؉/6؉ with DNA from 1,112 cervical scrapings. There was 79% overall agreement (kappa ‫؍‬ 0.816). BSGP5؉/6؉ was significantly more sensitive than GP5؉/6؉ for detection of HPV 30, 39, 42, 44, 51, 52, 53, 68, 73, and 82, detecting 212 additional HPV infections and increasing the proportion of multiple infections from 17.2 to 26.9% in cancer patients. In conclusion, BSGP5؉/6؉ multiplexed with ␤-globin PCR provides an improvement in type-specific amplification sensitivity and homogeneity compared to GP5؉/6؉ and offers simultaneous internal control of DNA quality. BSGP5؉/6؉-MPG, therefore, is suitable for epidemiologic and also diagnostic applications.High-risk human papillomavirus (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 (11). Until now, more than 100 HPV genotypes are fully characterized based on the isolation of complete genomes, with evidence that a larger number exist (6). There are 48 known mucosal HPV types, which are further divided into three groups based on their epidemiological association with cancer: HR types, including types 16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82; putative HR (pHR) types, including types 26, 53, and 66; and low-risk (LR) types, including types 6, 11, 40, 42, 43, 44, and 70 (11). HPV genotyping is of particular importance for studying the natural history of HPV infections and the diseases associated therewith, for monitoring vaccine efficacy, and for identifying novel HPV candidates for vaccine development. Further, it is required to diagnose multiple HPV infections.At present, there are several PCR-based HPV detection methods; one of the most commonly applied uses the GP5ϩ/6ϩ (GP) primer set (18), which targets conserved sequences within the L1 region of the virus genome flanking highly variable type...

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, broadspectrum 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 infectio...