Preclinical Evaluation of Two Real-Time, Reverse Transcription-PCR Assays for Detection of the Severe Acute Respiratory Syndrome Coronavirus
Identifieur interne : 000642 ( Pmc/Curation ); précédent : 000641; suivant : 000643Preclinical Evaluation of Two Real-Time, Reverse Transcription-PCR Assays for Detection of the Severe Acute Respiratory Syndrome Coronavirus
Auteurs : Adam M. Bressler [Géorgie (pays)] ; Frederick S. Nolte [Géorgie (pays)]Source :
- Journal of Clinical Microbiology [ 0095-1137 ] ; 2004.
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.
Url:
DOI: 10.1128/JCM.42.3.987-991.2004
PubMed: 15004042
PubMed Central: 356893
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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Clin Microbiol</journal-id><journal-id journal-id-type="publisher-id">jcm</journal-id><journal-title>Journal of Clinical Microbiology</journal-title><issn pub-type="ppub">0095-1137</issn><issn pub-type="epub">1098-660X</issn><publisher><publisher-name>American Society for Microbiology</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">15004042</article-id><article-id pub-id-type="pmc">356893</article-id><article-id pub-id-type="publisher-id">1641</article-id><article-id pub-id-type="doi">10.1128/JCM.42.3.987-991.2004</article-id><article-categories><subj-group subj-group-type="heading"><subject>Virology</subject></subj-group></article-categories><title-group><article-title>Preclinical Evaluation of Two Real-Time, Reverse Transcription-PCR Assays for Detection of the Severe Acute Respiratory Syndrome Coronavirus</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Bressler</surname><given-names>Adam M.</given-names></name><xref ref-type="aff" rid="aff0"></xref></contrib><contrib contrib-type="author"><name><surname>Nolte</surname><given-names>Frederick S.</given-names></name><xref ref-type="aff" rid="aff0"></xref><xref ref-type="corresp" rid="cor1">*</xref></contrib></contrib-group><aff id="aff0">Departments of Medicine (Infectious Diseases) and Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia</aff><author-notes><fn id="cor1"><label>*</label><p>Corresponding author. Mailing address: Emory University Hospital, Clinical Laboratories, Room F145, 1364 Clifton Rd., NE, Atlanta, GA 30322. Phone: (404) 712-7297. Fax: (404) 712-4632. E-mail: <email>fnolte@emory.edu</email>.</p></fn></author-notes><pub-date pub-type="ppub"><month>3</month><year>2004</year></pub-date><volume>42</volume><issue>3</issue><fpage>987</fpage><lpage>991</lpage><history><date date-type="received"><day>23</day><month>9</month><year>2003</year></date><date date-type="rev-recd"><day>27</day><month>10</month><year>2003</year></date><date date-type="accepted"><day>14</day><month>11</month><year>2003</year></date></history><copyright-statement>Copyright © 2004, American Society for Microbiology</copyright-statement><copyright-year>2004</copyright-year><abstract><p>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.</p></abstract></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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