Sequence variation of human papillomavirus type 16 and measurement of viral integration by quantitative PCR.

Given that the integration of human papillomavirus type 16 (HPV16) into the host genome occurs preferentially with the disruption of the E2 gene, a ratio of E2 to E7 gene copies is often used as a marker for integration. It is largely undetermined, however, whether ratio estimates are affected by HPV intratypic variations. We assembled four plasmid constructs, each containing a DNA fragment from an HPV16 European, Asian-American, African-1, or African-2 variant. These constructs and nine cervical swab samples were assayed by real-time PCR with two primer-probe sets for each gene: a specific set, fully complementary to the HPV16 prototype, and a degenerate set, incorporating degenerate bases at positions where nucleotides differed among the variants. The ratio of E2 to E7 gene copies for the European variant construct was close to 1, no matter which sets of primers and probes were used. While the ratios for the African-1 and Asian-American variant constructs remained close to 1 with the degenerate sets of primers and probes, the ratios were 0.36 and 2.57, respectively, with the specific sets of primers and probes. In addition, a nucleotide alteration at the position immediately following the 3' end of the E2 forward primer binding site was found to be responsible for an underestimation of E2 gene copies for the African-2 variant construct. Similar patterns were found in nine cervical samples. In conclusion, mismatches between the primers and probes and their targets due to HPV16 intratypic variations would introduce errors in testing for integration; this situation can be sufficiently ameliorated by incorporating degenerate bases into the primers and probes.