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 for HPV detection and typing was developed, taking advantage of two widely used pairs of human papillomavirus (HPV)-specific PCR primers, MY09/MY11 and GP5/GP6, and 33P-labeled dideoxynucleotides. In this study, 105 pathological specimens were examined: 89% were diagnosed as cervical intraepithelial neoplasia (CIN) grade I-III, 76.2% 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%-25% 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 L1 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 precision.
Feoli-Fonseca
J C
JC
Département de Pathologie, Centre de Recherche, Montréal, Québec, Canada.
Oligny
L L
LL
Filion
M
M
Brochu
P
P
Simard
P
P
Russo
P A
PA
Yotov
W V
WV
D001003:Anal Canal / Q000821:virology; D002294:Carcinoma, Squamous Cell / Q000821:virology; D002584:Cervix Uteri / Q000821:virology; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000032:analysis*; D015224:Dideoxynucleosides; D004825:Epiglottis / Q000821:virology; D005260:Female; D005814:Genes, Viral; D006801:Humans; D007830:Larynx / Q000821:virology; D009506:Nevus / Q000139:chemically induced; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D012878:Skin Neoplasms / Q000139:chemically induced; D014412:Tumor Virus Infections / Q000821:virology*; D002583:Uterine Cervical Neoplasms / Q000821:virology; D014609:Uvula / Q000821:virology; D014773:Virology / Q000379:methods*; D014844:Vulva / Q000821:virology; D002578:Uterine Cervical Dysplasia / Q000821:virology
D015344:DNA Probes, HPV; D004279:DNA, Viral; D015224:Dideoxynucleosides