DETECTION OF GENITAL HUMAN PAPILLOMAVIRUS BY SINGLE-TUBE NESTED PCR AND TYPE-SPECIFIC OLIGONUCLEOTIDE HYBRIDIZATION

Cervical cancer is, on a global scale, the second most common form of cancer in women. Development of cervical carcinoma is strongly associa ted with infection by certain types of human papillomavirus (HPV), To facilitate the detection and molecular typing of HPV in clinical sampl es, nested-PCR amplification systems were developed for regions of the E1 and L1 genes. The nested amplifications were performed in a single reaction tube, and shifting between inner and outer primer pairs was achieved by a two-phase amplification with different annealing tempera tures, This method eliminates cross-contamination between samples duri ng transfer from the first to the second amplification step. A set of type-specific oligonucleotide probes were designed for the El system a nd used to distinguish 19 genital HPV types, The sensitivities of our amplification systems compare favorably with that for the L1 system on the basis of the MY09-MY11 primer pair (M, M, Manos, Y.Ting, D.K. Wri ght, A.J. Lewis, T.R Broker, and S.M. Wolinsky, Cancer Cells 7:209-214 , 1989) and our systems can be used on materials such as HPV-infected cell lines, cytobrush samples, cancer biopsies, and recent as well as archival Papanicolaou (Pap) smears, The high sensitivity coupled with the effective elimination of contamination in the transfer between the two amplification steps of the nested PCR makes these systems suitabl e for research as well as clinical analyses.