Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens

AimsPersistent infection indicated by detection of human papillomavirus 16 (HPV-16) on repeat testing over a period of time poses the greatest cervical cancer risk. However, variants of HPV-16, HPV-31 and HPV-33 may share several short sequence homologies in the hypervariable L1 gene commonly targeted for HPV genotyping. The purpose of this study was to introduce a robust laboratory procedure to validate HPV-16 detected in clinical specimens, using the GenBank sequence database as the standard reference for genotyping.MethodsA nested PCR with two pairs of consensus primers was used to amplify the HPV DNA released in crude proteinase K digest of the cervicovaginal cells in liquid-based Papanicolaou cytology specimens. The positive nested PCR products were used for direct automated DNA sequencing.ResultsA 48-base sequence downstream of the GP5+ priming site, or a 34-base sequence upstream thereof, was needed for unequivocal validation of an HPV-16 isolate. Selection of a 45-base, or shorter, sequence immediately downstream of the GP5+ site for Basic Local Alignment Search Tool sequence analysis invariably led to ambiguous genotyping results.ConclusionsDNA sequence analysis may be used for differential genotyping of HPV-16, HPV-31 and HPV-33 in clinical specimens. However, selection of the signature sequence for Basic Local Alignment Search Tool algorithms is crucial to distinguish certain HPV-16 variants from other closely related HPV genotypes.