Homogeneous Amplification of Genital Human Alpha Papillomaviruses by PCR Using Novel Broad-Spectrum GP5+ and GP6+ Primers

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 β-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 β-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 β-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.