A Two-Tier Polymerase Chain Reaction Direct Sequencing Method for Detecting and Typing Human Papillomaviruses in Pathological Specimens

Abstract

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.

Human papillomavirus (HPV) study of 691 pathological specimens from Quebec by PCR-direct sequencing approach

“…The two-tier approach provided increased sensitivity for HPV detection. As described previously for 105 specimens [Feoli-Fonseca et al, 1998b], the sensitivity for primers GP5 /6 alone was 66%, My09/11 alone yielded 58% HPV positives, and the``two-tier'' system (GP5 / 6 , followed by My09/11 for the HPV negative samples) yielded 76% HPV positive results for the same samples.…”

“…The method was recently published [Feoli-Fonseca et al, 1998b] and is summarized below. All samples were subjected ®rst to PCR ampli®cation with the GP5 /GP6 primer pair.…”

Multiplex nested PCR (MNP) assay for the detection of 15 high risk genotypes of human papillomavirus

“…Sequencing products were interrogated by an Applied Biosystems 310 Sequence Analyzer. Resulting sequences were analyzed by a BLASTn search using Genbank (National Centre for Biotechnology Information, (http://www.ncbi.nlm.nih.gov/; Feoli-Fonseca et al, 1998, 2001Rady et al, 1993).…”

Papillary squamous intraepithelial lesions of the uterine cervix: human papillomavirus-dependent changes in cell cycle expression and cytologic features

“…Each polymerase chain reaction (PCR) amplification reaction was carried out in a total volume of 50 μL containing a PCR master mixture (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 800 μmol/L each of dATP, dCTP, dGTP, 600 μmol/L dUTP, 10 pmol of each primer set, 1.25 IU/L, AmpliTaq Gold DNA polymerase; Applied Biosystems, San Francisco, CA). General consensus primers MY09/GP6+ were used for the first PCR round to amplify the corresponding part of the HPV LI gene [7]. A nested PCR was then carried out with the primers GP5 +/GP6+ according to previously published protocols [8].…”