Aims: Persistent 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.
Methods: A nested polymerase chain reaction (PCR) with two pairs of consensus primers was used to amplify the HPV DNA released in crude proteinase K digestate of the cervicovaginal cells in liquid-based Papanicolaou cytology specimens. The positive nested PCR products were used for direct automated DNA sequencing.
Results: A 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 (BLAST) sequence analysis invariably led to ambiguous genotyping results.
Conclusions: DNA 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 BLAST algorithms is crucial to distinguish certain HPV-16 variants from other closely related HPV genotypes.