Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens
- Correspondence to Dr Sin Hang Lee, Department of Pathology, Milford Hospital, 300 Seaside Avenue, Milford, CT 06460, USA;
- Accepted 2 October 2009
- Published Online First 26 October 2009
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 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.
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 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 Basic Local Alignment Search Tool algorithms is crucial to distinguish certain HPV-16 variants from other closely related HPV genotypes.
- HPV-16 genotyping
- DNA sequencing
- signature DNA sequences
- gynaecological pathology
- tumour virology
Competing interests Dr Sin Hang Lee declares that he is a pathologist at Milford Hospital, Milford, Connecticut, USA, and the director of Milford Medical Laboratory. Dr Lee receives a fixed salary from the hospital which charges fees for cancer biopsies and HPV testing. Dr Lee is also the president and a shareholder of HiFi DNA Tech, LLC (www.hifidna.com), a company specialised in transferring the Sanger DNA sequencing technology to community hospital laboratories for DNA testing. Veronica S Vigliotti: None to declare. Dr Suri Pappu: None to declare.
Ethics approval Ethics approval was obtained from the Milford Hospital Institutional Review Board.
Patient consent Not obtained.
Provenance and peer review Not commissioned; externally peer reviewed.