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J Clin Pathol. 2010 Mar;63(3):235-9. doi: 10.1136/jcp.2009.069401. Epub 2009 Oct 26.

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

Author information

1
Milford Hospital, Milford, Connecticut 06460, USA. sinhang.lee@milfordhospital.org

Abstract

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.

PMID:
19858529
PMCID:
PMC2921263
DOI:
10.1136/jcp.2009.069401
[Indexed for MEDLINE]
Free PMC Article
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