Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens.
OBJECTIVE
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.
Milford Hospital, Milford, Connecticut 06460, USA. sinhang.lee@milfordhospital.org
Sin Hang
SH
Lee
Veronica S
VS
Vigliotti
Suri
S
Pappu
D000328:Adult; D000368:Aged; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D030561:Databases, Nucleic Acid; D005260:Female; D005838:Genotype; D052162:Human papillomavirus 16 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D030361:Papillomavirus Infections / Q000150:complications / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D002583:Uterine Cervical Neoplasms / Q000821:virology*
D004279:DNA, Viral