Homogeneous Amplification of Genital Human Alpha Papillomaviruses by Novel Broad Spectrum BSGP5+/6+ PCR

Research Program Infection and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany

^* To whom correspondence should be addressed. Email: markus.schmitt{at}dkfz.de.

	Abstract

HPV DNA detection and typing is 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 describe and validate novel broad-spectrum (BS) primers targeting the GP5+/6+ region. Addition of 8 upstream and 2 downstream BSGP5+/6+ 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 100ng cellular DNA was obtained. Additionally, we integrated an internal -globin PCR into both HPV PCR systems allowing simultaneous DNA quality control without affecting sensitivity of HPV detection. Furthermore, we describe 5 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, compared to standard GP5+/6+ was evaluated with DNA from 1017 cervical scrapings and 95 cervical cancer biopsies. There was 79% overall agreement (kappa=0.815). 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 of 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.