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00019606-199812000-00005ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 1998 Lippincott Williams & Wilkins, Inc.7December 1998 p 317–323A Two-Tier Polymerase Chain Reaction Direct Sequencing Method for Detecting and Typing Human Papillomaviruses in Pathological SpecimensOriginal Article: PDF OnlyFeoli-Fonseca, Juan Carlos M.D.M. sc.; Filion, Mario PhD.; Brochu, Pierre M.D.; Simard, Pierre M.D.; Luc, L. Oligny M.D.; Pierre, A. Russo M.D.; Wagner, V. Yotov M.D. Ph.D.Département de Pathologic (J.C.F., L.L.O., P.B., P.S., P.A.R., W.V.Y.). Département de Pédiatric (W.V.Y.). Centre de Recherche (P.A.R., W.V.Y.). Algène Biotechnologies (M.F.). Centre de Cancérologie Charles Bruneau W.V.Y.). Hôpital Ste-Justine. and Programme de Biologie Moléculaire (W.V.Y.). Faculté des Études Supérieures. Université de Montréal. Montréal. Québec, CanadaAbstractAn in-house polymerase chain reaction direct sequencing (PCR-DS) approach for HPV detection and typing was developed, taking advantage of two widely used pairs of human papillomavirus (HPV)-specific PCR primers, MY09/MYII and GP5/GP6, and 33P-labeled dideoxynucleotides. In this study, 105 pathological specimens were examined: 89|X% were diagnosed as cervical intraepithelial neoplasia (CIN) grade I-III, 76. 2|X% were HPV-positive by PCR-DS. The PCR using GP5/GP6 (first tier) and MY09/MY11 primers (second tier for the GP5/GP6-negative samples) detected additional 15|X%-25|X% HPV-positive samples compared with each pair used separately. Direct sequencing was then used to type the HPV. A readout of a sequence as short as 34 nucleotides within a specific region in the LI gene is sufficient to type known or novel sequences. Because of its high sensitivity and cost-effectiveness, the two-tier PCR-DS was adopted by the authors as the current method of choice for HPV diagnosis with ultimate sequence precisionHuman papillomavirus; Polymerase chain reaction; Direct sequencing; Cervical biopsy; Cervical intraepithelial neoplasia; Cervical intraepithelial neoplasia; MY09; GP5.A Two-Tier Polymerase Chain Reaction Direct Sequencing Method for Detecting and Typing Human Papillomaviruses in Pathological SpecimensFeoli-Fonseca Juan Carlos M.D.M. sc.; Filion, Mario PhD.; Brochu, Pierre M.D.; Simard, Pierre M.D.; Luc, L. Oligny M.D.; Pierre, A. Russo M.D.; Wagner, V. Yotov M.D. Ph.D.Original Article: PDF OnlyOriginal Article: PDF Only67p 317-323