A two-tier polymerase chain reaction direct sequencing method for detecting and typing human papillomaviruses in pathological specimens

An in-house polymerase chain reaction direct sequencing (PCR-DS) approach f or HPV detection and typing was developed, taking advantage of two widely u sed pairs of human papillomavirus (HPV)-specific PCR primers, MY09/MY11 and GP5/GP6, and P-33-labeled dideoxynucleotides. In this study, 105 pathologi cal specimens were examined: 89% were diagnosed as cervical intraepithelial neoplasia (CIN) grade IIII, 76.2% were HPV-positive by PCR-DS. The PCR usi ng GP5/GP6 (first tier) and MY09/MY11 primers (second tier for the GP5/GP6- negative samples) detected additional 15%-25% HPV-positive samples compared with each pair used separately. Direct sequencing was then used to type th e 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. Beca use 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 w ith ultimate sequence precision.