SrasV1, Main, Exploration, bibRecord, 005B40

Evaluation of advanced reverse transcription-PCR assays and an alternative PCR target region for detection of severe acute respiratory syndrome-associated coronavirus

Identifieur interne : 005B40 ( Main/Exploration ); précédent : 005B39; suivant : 005B41

Evaluation of advanced reverse transcription-PCR assays and an alternative PCR target region for detection of severe acute respiratory syndrome-associated coronavirus

Auteurs : Christian Drosten [Allemagne] ; Lily-Lily Chiu [Singapour] ; Marcus Panning [Allemagne] ; HOE NAM LEONG [Singapour] ; Wolfgang Preiser [Allemagne] ; John S. Tam [Hong Kong] ; Stephan Günther [Allemagne] ; Stefanie Kramme [Allemagne] ; Petra Emmerich [Allemagne] ; WOOI LOON NG [Singapour] ; Herbert Schmitz [Allemagne] ; Evelyn S. C. Koay [Singapour]

Source :

Journal of clinical microbiology : (Print) [ 0095-1137 ] ; 2004.

RBID : Pascal:04-0283719

Descripteurs français

KwdFr :
- ARN viral (génétique), ARN viral (isolement et purification), Amorces ADN (génétique), Faux négatifs, Gènes viraux, Humains, Nucléocapside (génétique), RNA replicase (génétique), RT-PCR (), Syndrome respiratoire aigu sévère (diagnostic), Syndrome respiratoire aigu sévère (virologie), Séquence nucléotidique, Virus du SRAS (génétique), Virus du SRAS (isolement et purification), Études rétrospectives.
MESH :
- diagnostic : Syndrome respiratoire aigu sévère.
- génétique : ARN viral, Amorces ADN, Nucléocapside, RNA replicase, Virus du SRAS.
- isolement et purification : ARN viral, Virus du SRAS.
- virologie : Syndrome respiratoire aigu sévère.
Pascal (Inist)
- Coronavirus, Faux négatifs, Gènes viraux, Humains, RT-PCR, Réaction chaîne polymérase RT, Réaction chaîne polymérase, Détection, Microbiologie, Syndrome respiratoire aigu sévère, Séquence nucléotidique, Études rétrospectives.

English descriptors

KwdEn :
- Base Sequence, Coronavirus, DNA Primers (genetics), Detection, False Negative Reactions, Genes, Viral, Humans, Microbiology, Nucleocapsid (genetics), Polymerase chain reaction, RNA Replicase (genetics), RNA, Viral (genetics), RNA, Viral (isolation & purification), Retrospective Studies, Reverse Transcriptase Polymerase Chain Reaction (methods), Reverse Transcriptase Polymerase Chain Reaction (statistics & numerical data), Reverse transcription polymerase chain reaction, SARS Virus (genetics), SARS Virus (isolation & purification), Severe Acute Respiratory Syndrome (diagnosis), Severe Acute Respiratory Syndrome (virology), Severe acute respiratory syndrome.
MESH :
- chemical , genetics : DNA Primers, RNA Replicase, RNA, Viral.
- diagnosis : Severe Acute Respiratory Syndrome.
- genetics : Nucleocapsid, SARS Virus.
- chemical , isolation & purification : RNA, Viral, SARS Virus.
- methods : Reverse Transcriptase Polymerase Chain Reaction.
- statistics & numerical data : Reverse Transcriptase Polymerase Chain Reaction.
- virology : Severe Acute Respiratory Syndrome.
- Base Sequence, False Negative Reactions, Genes, Viral, Humans, Retrospective Studies.

Abstract

First-generation reverse transcription-PCR (RT-PCR) assays for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) gave false-negative results in a considerable fraction of patients. In the present study, we evaluated two second-generation, replicase (R) gene-based, real-time RT-PCR test kits-the RealArt HPA coronavirus LC kit (Artus, Hamburg, Germany) and the LightCycler SARS-CoV quantification kit (Roche, Penzberg, Germany)-and a real-time RT-PCR assay for the nucleocapsid (N) gene. Detecting the N-gene RNA might be advantageous due to its high abundance in cells. The kits achieved sensitivities of 70.8% (Artus) and 67.1% (Roche) in 66 specimens from patients with confirmed SARS (samples primarily from the upper and lower respiratory tract and stool). The sensitivity of the N-gene assay was 74.2%. The differences in all of the sensitivities were not statistically significant (P = 0.680 [analysis of variance]). Culture cells initially contained five times more N- than R-gene RNA, but the respective levels converged during 4 days of virus replication. In clinical samples the median concentrations of R- and N-gene RNA, respectively, were 1.2 x 10⁶ and 2.8 x 10⁶ copies/ml (sputum and endotracheal aspirates), 4.3 x 10⁴ and 5.5 x 10⁴ copies/ml (stool), and 5.5 x 10² and 5.2 x 10² copies/sample (throat swabs and saliva). Differences between the samples types were significant but not between the types of target RNA. All (n = 12) samples from the lower respiratory tract tested positive in all tests. In conclusion, the novel assays are more sensitive than the first-generation tests, but they still do not allow a comprehensive ruling out of SARS. Methods for the routine sampling of sputum without infection risk are needed to improve SARS RT-PCR.

Url:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC404639

Affiliations:

Le document en format XML

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<author><name sortKey="Wooi Loon Ng" sort="Wooi Loon Ng" uniqKey="Wooi Loon Ng" last="Wooi Loon Ng">WOOI LOON NG</name>
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<author><name sortKey="Schmitz, Herbert" sort="Schmitz, Herbert" uniqKey="Schmitz H" first="Herbert" last="Schmitz">Herbert Schmitz</name>
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<s3>DEU</s3>
<sZ>1 aut.</sZ>
<sZ>3 aut.</sZ>
<sZ>7 aut.</sZ>
<sZ>8 aut.</sZ>
<sZ>9 aut.</sZ>
<sZ>11 aut.</sZ>
</inist:fA14>
<country>Allemagne</country>
<placeName><settlement type="city">Hambourg</settlement>
<region type="land" nuts="2">Hambourg</region>
</placeName>
</affiliation>
</author>
<author><name sortKey="Koay, Evelyn S C" sort="Koay, Evelyn S C" uniqKey="Koay E" first="Evelyn S. C." last="Koay">Evelyn S. C. Koay</name>
<affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital</s1>
<s3>SGP</s3>
<sZ>2 aut.</sZ>
<sZ>10 aut.</sZ>
<sZ>12 aut.</sZ>
</inist:fA14>
<country>Singapour</country>
<wicri:noRegion>Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital</wicri:noRegion>
</affiliation>
<affiliation wicri:level="4"><inist:fA14 i1="06"><s1>Department of Pathology, National University of Singapore</s1>
<s3>SGP</s3>
<sZ>10 aut.</sZ>
<sZ>12 aut.</sZ>
</inist:fA14>
<country>Singapour</country>
<orgName type="university">Université nationale de Singapour</orgName>
</affiliation>
</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>Base Sequence</term>
<term>Coronavirus</term>
<term>DNA Primers (genetics)</term>
<term>Detection</term>
<term>False Negative Reactions</term>
<term>Genes, Viral</term>
<term>Humans</term>
<term>Microbiology</term>
<term>Nucleocapsid (genetics)</term>
<term>Polymerase chain reaction</term>
<term>RNA Replicase (genetics)</term>
<term>RNA, Viral (genetics)</term>
<term>RNA, Viral (isolation & purification)</term>
<term>Retrospective Studies</term>
<term>Reverse Transcriptase Polymerase Chain Reaction (methods)</term>
<term>Reverse Transcriptase Polymerase Chain Reaction (statistics & numerical data)</term>
<term>Reverse transcription polymerase chain reaction</term>
<term>SARS Virus (genetics)</term>
<term>SARS Virus (isolation & purification)</term>
<term>Severe Acute Respiratory Syndrome (diagnosis)</term>
<term>Severe Acute Respiratory Syndrome (virology)</term>
<term>Severe acute respiratory syndrome</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><term>ARN viral (génétique)</term>
<term>ARN viral (isolement et purification)</term>
<term>Amorces ADN (génétique)</term>
<term>Faux négatifs</term>
<term>Gènes viraux</term>
<term>Humains</term>
<term>Nucléocapside (génétique)</term>
<term>RNA replicase (génétique)</term>
<term>RT-PCR ()</term>
<term>Syndrome respiratoire aigu sévère (diagnostic)</term>
<term>Syndrome respiratoire aigu sévère (virologie)</term>
<term>Séquence nucléotidique</term>
<term>Virus du SRAS (génétique)</term>
<term>Virus du SRAS (isolement et purification)</term>
<term>Études rétrospectives</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA Primers</term>
<term>RNA Replicase</term>
<term>RNA, Viral</term>
</keywords>
<keywords scheme="MESH" qualifier="diagnosis" xml:lang="en"><term>Severe Acute Respiratory Syndrome</term>
</keywords>
<keywords scheme="MESH" qualifier="diagnostic" xml:lang="fr"><term>Syndrome respiratoire aigu sévère</term>
</keywords>
<keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Nucleocapsid</term>
<term>SARS Virus</term>
</keywords>
<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ARN viral</term>
<term>Amorces ADN</term>
<term>Nucléocapside</term>
<term>RNA replicase</term>
<term>Virus du SRAS</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>RNA, Viral</term>
<term>SARS Virus</term>
</keywords>
<keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>ARN viral</term>
<term>Virus du SRAS</term>
</keywords>
<keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Reverse Transcriptase Polymerase Chain Reaction</term>
</keywords>
<keywords scheme="MESH" qualifier="statistics & numerical data" xml:lang="en"><term>Reverse Transcriptase Polymerase Chain Reaction</term>
</keywords>
<keywords scheme="MESH" qualifier="virologie" xml:lang="fr"><term>Syndrome respiratoire aigu sévère</term>
</keywords>
<keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Severe Acute Respiratory Syndrome</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Base Sequence</term>
<term>False Negative Reactions</term>
<term>Genes, Viral</term>
<term>Humans</term>
<term>Retrospective Studies</term>
</keywords>
<keywords scheme="Pascal" xml:lang="fr"><term>Coronavirus</term>
<term>Faux négatifs</term>
<term>Gènes viraux</term>
<term>Humains</term>
<term>RT-PCR</term>
<term>Réaction chaîne polymérase RT</term>
<term>Réaction chaîne polymérase</term>
<term>Détection</term>
<term>Microbiologie</term>
<term>Syndrome respiratoire aigu sévère</term>
<term>Séquence nucléotidique</term>
<term>Études rétrospectives</term>
</keywords>
</textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en">First-generation reverse transcription-PCR (RT-PCR) assays for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) gave false-negative results in a considerable fraction of patients. In the present study, we evaluated two second-generation, replicase (R) gene-based, real-time RT-PCR test kits-the RealArt HPA coronavirus LC kit (Artus, Hamburg, Germany) and the LightCycler SARS-CoV quantification kit (Roche, Penzberg, Germany)-and a real-time RT-PCR assay for the nucleocapsid (N) gene. Detecting the N-gene RNA might be advantageous due to its high abundance in cells. The kits achieved sensitivities of 70.8% (Artus) and 67.1% (Roche) in 66 specimens from patients with confirmed SARS (samples primarily from the upper and lower respiratory tract and stool). The sensitivity of the N-gene assay was 74.2%. The differences in all of the sensitivities were not statistically significant (P = 0.680 [analysis of variance]). Culture cells initially contained five times more N- than R-gene RNA, but the respective levels converged during 4 days of virus replication. In clinical samples the median concentrations of R- and N-gene RNA, respectively, were 1.2 x 10<sup>6</sup>
 and 2.8 x 10<sup>6</sup>
 copies/ml (sputum and endotracheal aspirates), 4.3 x 10<sup>4</sup>
 and 5.5 x 10<sup>4</sup>
 copies/ml (stool), and 5.5 x 10<sup>2</sup>
 and 5.2 x 10<sup>2</sup>
 copies/sample (throat swabs and saliva). Differences between the samples types were significant but not between the types of target RNA. All (n = 12) samples from the lower respiratory tract tested positive in all tests. In conclusion, the novel assays are more sensitive than the first-generation tests, but they still do not allow a comprehensive ruling out of SARS. Methods for the routine sampling of sputum without infection risk are needed to improve SARS RT-PCR.</div>
</front>
</TEI>
<affiliations><list><country><li>Allemagne</li>
<li>Hong Kong</li>
<li>Singapour</li>
</country>
<region><li>District de Darmstadt</li>
<li>Hambourg</li>
<li>Hesse (Land)</li>
</region>
<settlement><li>Francfort-sur-le-Main</li>
<li>Hambourg</li>
<li>Sha Tin</li>
</settlement>
<orgName><li>Université chinoise de Hong Kong</li>
<li>Université nationale de Singapour</li>
</orgName>
</list>
<tree><country name="Allemagne"><region name="Hambourg"><name sortKey="Drosten, Christian" sort="Drosten, Christian" uniqKey="Drosten C" first="Christian" last="Drosten">Christian Drosten</name>
</region>
<name sortKey="Emmerich, Petra" sort="Emmerich, Petra" uniqKey="Emmerich P" first="Petra" last="Emmerich">Petra Emmerich</name>
<name sortKey="Gunther, Stephan" sort="Gunther, Stephan" uniqKey="Gunther S" first="Stephan" last="Günther">Stephan Günther</name>
<name sortKey="Kramme, Stefanie" sort="Kramme, Stefanie" uniqKey="Kramme S" first="Stefanie" last="Kramme">Stefanie Kramme</name>
<name sortKey="Panning, Marcus" sort="Panning, Marcus" uniqKey="Panning M" first="Marcus" last="Panning">Marcus Panning</name>
<name sortKey="Preiser, Wolfgang" sort="Preiser, Wolfgang" uniqKey="Preiser W" first="Wolfgang" last="Preiser">Wolfgang Preiser</name>
<name sortKey="Schmitz, Herbert" sort="Schmitz, Herbert" uniqKey="Schmitz H" first="Herbert" last="Schmitz">Herbert Schmitz</name>
</country>
<country name="Singapour"><noRegion><name sortKey="Chiu, Lily Lily" sort="Chiu, Lily Lily" uniqKey="Chiu L" first="Lily-Lily" last="Chiu">Lily-Lily Chiu</name>
</noRegion>
<name sortKey="Hoe Nam Leong" sort="Hoe Nam Leong" uniqKey="Hoe Nam Leong" last="Hoe Nam Leong">HOE NAM LEONG</name>
<name sortKey="Koay, Evelyn S C" sort="Koay, Evelyn S C" uniqKey="Koay E" first="Evelyn S. C." last="Koay">Evelyn S. C. Koay</name>
<name sortKey="Koay, Evelyn S C" sort="Koay, Evelyn S C" uniqKey="Koay E" first="Evelyn S. C." last="Koay">Evelyn S. C. Koay</name>
<name sortKey="Wooi Loon Ng" sort="Wooi Loon Ng" uniqKey="Wooi Loon Ng" last="Wooi Loon Ng">WOOI LOON NG</name>
<name sortKey="Wooi Loon Ng" sort="Wooi Loon Ng" uniqKey="Wooi Loon Ng" last="Wooi Loon Ng">WOOI LOON NG</name>
</country>
<country name="Hong Kong"><noRegion><name sortKey="Tam, John S" sort="Tam, John S" uniqKey="Tam J" first="John S." last="Tam">John S. Tam</name>
</noRegion>
</country>
</tree>
</affiliations>
</record>

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Evaluation of advanced reverse transcription-PCR assays and an alternative PCR target region for detection of severe acute respiratory syndrome-associated coronavirus

Evaluation of advanced reverse transcription-PCR assays and an alternative PCR target region for detection of severe acute respiratory syndrome-associated coronavirus

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