Document Detail


Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples.
MedLine Citation:
PMID:  16751488     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
M Isabel Costafreda; Albert Bosch; Rosa M Pintó
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Applied and environmental microbiology     Volume:  72     ISSN:  0099-2240     ISO Abbreviation:  Appl. Environ. Microbiol.     Publication Date:  2006 Jun 
Date Detail:
Created Date:  2006-06-05     Completed Date:  2006-08-30     Revised Date:  2013-06-07    
Medline Journal Info:
Nlm Unique ID:  7605801     Medline TA:  Appl Environ Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  3846-55     Citation Subset:  IM    
Affiliation:
Department of Microbiology, School of Biology, University of Barcelona, Diagonal 645, 08028 Barcelona, Spain.
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MeSH Terms
Descriptor/Qualifier:
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:
0/RNA, Viral
Comments/Corrections

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