Denaturing high-performance liquid chromatography for detecting and typing genital human papillomavirus

Human papillomaviruses (HPVs) are important in the development of human cancers, including cervical and oral tumors. However, most existing methods for HPV typing cannot routinely distinguish among the more than 100 distinct types of HPV or the natural HPV intratypic variants that have also been documented. To address this problem, we developed a novel method, general primer-denaturing high-performance liquid chromatography (GP-dHPLC), for the detection and typing of genital HPV using an automated 96-well plate format. GP-dHPLC uses general primer PCR (GP-PCR) to amplify the viral DNA and then analyzes the GP-PCR products by denaturing high-performance liquid chromatography (dHPLC). A number of different primer pairs with homology to most known genital HPV types were tested, and the L1C1-L1C2M pair specific for the L1 region of the viral genome was chosen. A set of HPV standard control patterns, consisting of those for HPV types 16, 18, 31, 33, 39, 45, 51, 52, 56, 58, 59, 6, and 11, was established for genital HPV typing. One hundred eighty-six frozen and formalin-fixed cervical cancer tissue samples were analyzed for the presence of HPV and the HPV type by this method, and 95.8% of them were found to contain HPV DNA. GP-dHPLC accurately discriminated among HPV variants that differed by as little as one nucleotide. Several new variants of HPV types 16, 18, 39, 45, 52, and 59 were identified. Moreover, multiple HPV infections were detected in 26.6% of the samples. Our results indicate that HPV typing by GP-dHPLC permits discrimination of common genital HPV types, detection of multiple HPV infections, and identification of HPV variants in clinical samples.