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

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 targ...

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Autores principales: Lee, Sin Hang, Vigliotti, Veronica S, Pappu, Suri
Formato: Texto
Lenguaje:English
Publicado: BMJ Group 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2921263/
https://www.ncbi.nlm.nih.gov/pubmed/19858529
http://dx.doi.org/10.1136/jcp.2009.069401
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author Lee, Sin Hang
Vigliotti, Veronica S
Pappu, Suri
author_facet Lee, Sin Hang
Vigliotti, Veronica S
Pappu, Suri
author_sort Lee, Sin Hang
collection PubMed
description 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.
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spelling pubmed-29212632010-08-17 Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens Lee, Sin Hang Vigliotti, Veronica S Pappu, Suri J Clin Pathol Original Article 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. BMJ Group 2009-10-26 2010-03 /pmc/articles/PMC2921263/ /pubmed/19858529 http://dx.doi.org/10.1136/jcp.2009.069401 Text en © 2009, Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited, the use is non commercial and is otherwise in compliance with the license. See: http://creativecommons.org/licenses/by-nc/2.0/ and http://creativecommons.org/licenses/by-nc/2.0/legalcode.
spellingShingle Original Article
Lee, Sin Hang
Vigliotti, Veronica S
Pappu, Suri
Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens
title Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens
title_full Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens
title_fullStr Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens
title_full_unstemmed Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens
title_short Signature sequence validation of human papillomavirus type 16 (HPV-16) in clinical specimens
title_sort signature sequence validation of human papillomavirus type 16 (hpv-16) in clinical specimens
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2921263/
https://www.ncbi.nlm.nih.gov/pubmed/19858529
http://dx.doi.org/10.1136/jcp.2009.069401
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