The Use of General Primers in the Polymerase Chain Reaction Permits the Detection of a Broad Spectrum of Human Papillomavirus Genotypes

TLDR

A novel PCR method was developed that permits detection of 11 different HPV genotypes using two general primer sets. The method uses two general primer pairs selected from the conserved L1 region, tested on cloned HPV genotypes, with specificity confirmed by blot hybridization and RsaI digestion. The assay tolerates up to three primer mismatches, detects 11 HPV genotypes—including HPV‑1a, ‑6, ‑8, ‑11, ‑13, ‑16, ‑18, ‑30, ‑31, ‑32, ‑33—at sub‑picogram levels in cloned DNA, cell lines, and clinical samples, making it a powerful tool for identifying novel HPV genotypes in dysplasias and squamous cell carcinomas.

Abstract

A novel polymerase chain reaction (PCR) method was developed that permits the detection of 11 different human papillomavirus (HPV) genotypes using two general primer sets. By computer-assisted sequence analysis, two pairs of general primers were selected from the conserved L1 open reading frame and tested in the PCR on a set of cloned HPV genotypes. Experimental analysis showed that up to three mismatches between primers and target DNA did not influence the efficiency of the assay. The use of these primers in the PCR enabled the detection of HPV genotypes HPV-1a, -6, -8, -11, -13, -16, -18, -30, -31, -32 and -33, and was also successfully applied to well characterized cervical carcinoma cell lines and clinical samples. For the HPV types tested sub-picogram amounts of cloned DNA could be detected after general primer-mediated PCR and subsequent hybridization. The specificity of the amplification products was confirmed by blot hybridization procedures and RsaI restriction enzyme digestion. The results indicate that this PCR method can be a powerful tool for identifying novel HPV genotypes in dysplasias and squamous cell carcinomas suspected of having an HPV aetiology.