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

Lee SH, Vigliotti VS, Pappu S - J. Clin. Pathol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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Related in: MedlinePlus

This is a less than perfect computer-generated electropherogram similar to that shown in figure 1. An imperfect base-calling sequence tracing may prevent selection of a long enough sequence inclusive of the three crucial bases GTT (underlined) for Basic Local Alignment Search Tool (BLAST) algorithm, causing ambiguity in differential genotyping of human papillomavirus 16.
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Related In: Results  -  Collection

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fig3: This is a less than perfect computer-generated electropherogram similar to that shown in figure 1. An imperfect base-calling sequence tracing may prevent selection of a long enough sequence inclusive of the three crucial bases GTT (underlined) for Basic Local Alignment Search Tool (BLAST) algorithm, causing ambiguity in differential genotyping of human papillomavirus 16.

Mentions: Using the crude proteinase K digest of clinical materials of a complex nature for PCR and direct automated DNA sequencing might generate electropherograms of varying qualities. When the quality of an electropherogram was high, recognition of the 3-base ‘crucial’ sequence (figure 1) for differential genotyping of HPV-16 was straightforward by selecting at least 48 bases downstream of the GP5+ priming site for a BLAST analysis. However, when the electropherogram was less than perfect (figure 3), an inexperienced sequence analyst might have selected the first 34 bases for BLAST sequence alignment algorithms, leading to genotyping ambiguities or errors. For the latter reason, a GP6/MY11 heminested or an equivalent HiFi nested PCR primer amplicon was used to generate an extended electropherogram with an additional base-calling stretch upstream of the GP5+ priming site. In this upstream stretch, the GP5+ priming site became part of the sequence useful for base calling.


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

Lee SH, Vigliotti VS, Pappu S - J. Clin. Pathol. (2009)

This is a less than perfect computer-generated electropherogram similar to that shown in figure 1. An imperfect base-calling sequence tracing may prevent selection of a long enough sequence inclusive of the three crucial bases GTT (underlined) for Basic Local Alignment Search Tool (BLAST) algorithm, causing ambiguity in differential genotyping of human papillomavirus 16.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2921263&req=5

fig3: This is a less than perfect computer-generated electropherogram similar to that shown in figure 1. An imperfect base-calling sequence tracing may prevent selection of a long enough sequence inclusive of the three crucial bases GTT (underlined) for Basic Local Alignment Search Tool (BLAST) algorithm, causing ambiguity in differential genotyping of human papillomavirus 16.
Mentions: Using the crude proteinase K digest of clinical materials of a complex nature for PCR and direct automated DNA sequencing might generate electropherograms of varying qualities. When the quality of an electropherogram was high, recognition of the 3-base ‘crucial’ sequence (figure 1) for differential genotyping of HPV-16 was straightforward by selecting at least 48 bases downstream of the GP5+ priming site for a BLAST analysis. However, when the electropherogram was less than perfect (figure 3), an inexperienced sequence analyst might have selected the first 34 bases for BLAST sequence alignment algorithms, leading to genotyping ambiguities or errors. For the latter reason, a GP6/MY11 heminested or an equivalent HiFi nested PCR primer amplicon was used to generate an extended electropherogram with an additional base-calling stretch upstream of the GP5+ priming site. In this upstream stretch, the GP5+ priming site became part of the sequence useful for base calling.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

Show MeSH
Related in: MedlinePlus