The use of general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR

TLDR

The study aimed to generate elongated GP5+ and GP6+ primers by adding conserved sequences to the 3′ ends of GP5/6 to enhance HPV detection by PCR. The authors extended the 3′ ends of GP5 and GP6 with adjacent highly conserved sequences to create the GP5+ and GP6+ primers. The GP5+/6+ PCR assay achieved 10–100‑fold higher sensitivity than GP5/6, detected HPV at femtogram to picogram levels, increased overall positivity from 39 % to 43 % in cervical samples, and identified additional uncommon HPV types missed by the original primers.

Abstract

Sequence analysis of human papillomavirus (HPV) general primer GP5/6 mediated PCR products revealed the presence of short highly conserved sequences adjacent to the 3′ ends of both primers. Part of these sequences was used to elongate GP5 and GP6 at their 3′ ends to generate the primers GP5+ and GP6+, respectively. Compared with the GP5/6 PCR, GP5+/6+ specific PCR on 22 cloned mucosotropic HPVs revealed an improved HPV detection, reflected by a 10- to 100-fold higher sensitivity and a markedly increased signal to background ratio, especially at the gel level. As determined on purified DNA, the sensitivity of this GP5+/6+ based assay was at the femtogram level for those HPV genotypes which match strongly with the primers (e.g. HPV-16) and at the picogram level for HPV types (e.g. HPV-39 and -51) having four or more mismatches with one or both primers. Application of both methods on 264 cervical scrapes of a cohort of women participating in a prospective follow-up study revealed an increase of total HPV positivity from 39% (GP5/6 PCR) to 43% (GP5+/6+PCR) of the scrapes. Additional HPV typing by PCR specific for the HPV-6, -11, -16, -18, -31 and -33 revealed that all GP5+/6+ PCR positive cases which were negative by GP5/6 PCR (n = 12) contained HPV types different from these six types. These data indicate that the GP5+/6+ PCR method provides an increased detection level mainly of uncommon, apparently poorly matched HPV types in cervical scrapes and most likely in the enlargement of the spectrum of HPVs detectable by this assay.