| Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples. | |
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MedLine Citation:
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PMID: 16751488 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5' noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness. |
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Authors:
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M Isabel Costafreda; Albert Bosch; Rosa M Pintó |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Applied and environmental microbiology Volume: 72 ISSN: 0099-2240 ISO Abbreviation: Appl. Environ. Microbiol. Publication Date: 2006 Jun |
Date Detail:
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Created Date: 2006-06-05 Completed Date: 2006-08-30 Revised Date: 2013-06-07 |
Medline Journal Info:
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Nlm Unique ID: 7605801 Medline TA: Appl Environ Microbiol Country: United States |
Other Details:
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Languages: eng Pagination: 3846-55 Citation Subset: IM |
Affiliation:
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Department of Microbiology, School of Biology, University of Barcelona, Diagonal 645, 08028 Barcelona, Spain. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Base Sequence Consensus Sequence Hepatitis A virus / genetics, isolation & purification* Humans Molecular Sequence Data RNA, Viral / genetics, isolation & purification Reproducibility of Results Reverse Transcriptase Polymerase Chain Reaction / methods* Shellfish / virology* |
| Chemical | |
Reg. No./Substance:
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0/RNA, Viral |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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