A novel strategy for human papillomavirus detection and genotyping with SybrGreen and molecular beacon polymerase chain reaction

Human papillomaviruses (HPVs) play an important role in the pathogenesis of cervical cancer. For identification of the large number of different HPV t ypes found in (pre)malignant lesions, a robust methodology is needed that c ombines general HPV detection with HPV genotyping. We have developed for fo rmaldehyde-fixed samples a strategy that, in a homogenous, real-time fluore scence polymerase chain reaction (PCR)-based assay, accomplishes general HP V detection by SybrGreen reporting of HPV-DNA amplicons, and genotyping of seven prevalent HPV types (Hpv-6, -11, -16, -is, -31, -33, -45) by real-tim e molecular beacon PCR. The false-positive rate of the HPV SybrGreen-PCR wa s 4%, making it well suited as a prescreening, general HPV detection techno logy. The type specificity of the seven selected BW molecular beacons was 1 00% and double infections were readily identified. The multiplexing capacit y of the HPV molecular beacon PCR was analyzed and up to three differently labeled molecular beacons could be used in one PCR reaction without observi ng cross talk. The inherent quantitation capacities of real-time fluorescen ce PCR allowed the determination of average HPV copy number per cell. We co nclude that the HPV SybrGreen-PCR in combination with the HPV molecular bea con PCR provides a robust, sensitive, and quantitative general HPV detectio n and genotyping methodology.