Preclinical evaluation of two real-time, reverse transcription-PCR assays for detection of the severe acute respiratory syndrome coronavirus
Identifieur interne : 000798 ( PascalFrancis/Checkpoint ); précédent : 000797; suivant : 000799Preclinical evaluation of two real-time, reverse transcription-PCR assays for detection of the severe acute respiratory syndrome coronavirus
Auteurs : Adam M. Bressler [États-Unis] ; Frederick S. NolteSource :
- Journal of clinical microbiology : (Print) [ 0095-1137 ] ; 2004.
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Abstract
We verified the analytical performance characteristics of a previously described real-time reverse transcription-PCR (RT-PCR) assay targeting the open reading frame (ORF) 1b region of the severe acute respiratory syndrome coronavirus (SARS-CoV) with RNA transcripts. We then compared it to a novel nucleocapsid gene real-time RT-PCR assay with genomic RNA. The assays differed only in the primer and probe sequences and final concentrations. A commercially available armored RNA (Ambion, Austin, Tex.) was evaluated as positive control for the ORF 1b assay. The analytical sensitivity, reproducibility, amplification efficiency, and dynamic range of the assays were similar. Both were specific for SARS-CoV as determined by testing against human CoV 229E and OC43, specimens from patients without SARS, and by BLAST searches of GenBank for primer and probe sequence homology. The armored RNA was found to be a suitable positive control for the ORF 1b assay that could be reliably recovered and amplified from a variety of clinical specimens.
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Bressler</name><affiliation wicri:level="2"><inist:fA14 i1="01"><s1>Departments of Medicine (Infectious Diseases) and Pathology and Laboratory Medicine, Emory University School of Medicine</s1><s2>Atlanta, Georgia</s2><s3>USA</s3></inist:fA14><country>États-Unis</country><placeName><region type="state">Géorgie (États-Unis)</region></placeName></affiliation></author><author><name sortKey="Nolte, Frederick S" sort="Nolte, Frederick S" uniqKey="Nolte F" first="Frederick S." last="Nolte">Frederick S. Nolte</name></author></analytic><series><title level="j" type="main">Journal of clinical microbiology : (Print)</title><title level="j" type="abbreviated">J. clin. microbiol. : (Print)</title><idno type="ISSN">0095-1137</idno><imprint><date when="2004">2004</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Journal of clinical microbiology : (Print)</title><title level="j" type="abbreviated">J. clin. microbiol. : (Print)</title><idno type="ISSN">0095-1137</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Coronavirus</term><term>Detection</term><term>Microbiology</term><term>Real time</term><term>Reverse transcription polymerase chain reaction</term><term>Severe acute respiratory syndrome</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Coronavirus</term><term>Temps réel</term><term>Réaction chaîne polymérase RT</term><term>Détection</term><term>Microbiologie</term><term>Syndrome respiratoire aigu sévère</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">We verified the analytical performance characteristics of a previously described real-time reverse transcription-PCR (RT-PCR) assay targeting the open reading frame (ORF) 1b region of the severe acute respiratory syndrome coronavirus (SARS-CoV) with RNA transcripts. We then compared it to a novel nucleocapsid gene real-time RT-PCR assay with genomic RNA. The assays differed only in the primer and probe sequences and final concentrations. A commercially available armored RNA (Ambion, Austin, Tex.) was evaluated as positive control for the ORF 1b assay. The analytical sensitivity, reproducibility, amplification efficiency, and dynamic range of the assays were similar. Both were specific for SARS-CoV as determined by testing against human CoV 229E and OC43, specimens from patients without SARS, and by BLAST searches of GenBank for primer and probe sequence homology. 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Nolte</name></noCountry><country name="États-Unis"><region name="Géorgie (États-Unis)"><name sortKey="Bressler, Adam M" sort="Bressler, Adam M" uniqKey="Bressler A" first="Adam M." last="Bressler">Adam M. Bressler</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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