Comparison of 9 different PCR primers for the rapid detection of severe acute respiratory syndrome coronavirus using 2 RNA extraction methods
Identifieur interne : 004E55 ( Main/Exploration ); précédent : 004E54; suivant : 004E56Comparison of 9 different PCR primers for the rapid detection of severe acute respiratory syndrome coronavirus using 2 RNA extraction methods
Auteurs : Linda Chui [Canada] ; Michael Drebot [Canada] ; Anton Andonov [Canada] ; Astrid Petrich [Canada] ; Martin Glushek [Canada] ; James Mahony [Canada]Source :
- Diagnostic microbiology and infectious disease [ 0732-8893 ] ; 2005.
Descripteurs français
- KwdFr :
- ARN viral (analyse), Amorces ADN (), Animaux, Cellules Vero, Sensibilité et spécificité, Syndrome respiratoire aigu sévère (virologie), Techniques d'amplification d'acides nucléiques (instrumentation), Trousses de réactifs pour diagnostic, Virus du SRAS (génétique), Virus du SRAS (isolement et purification).
- MESH :
- analyse : ARN viral.
- génétique : Virus du SRAS.
- isolement et purification : Virus du SRAS.
- virologie : Syndrome respiratoire aigu sévère.
- Pascal (Inist)
- Amorces ADN, Animaux, Cellules Vero, Coronavirus, Réaction chaîne polymérase, Détection, Méthode, Sensibilité et spécificité, Techniques d'amplification d'acides nucléiques, Temps réel, Réaction chaîne polymérase RT, Sensibilité, Spécificité, Microbiologie, Syndrome respiratoire aigu sévère, Trousses de réactifs pour diagnostic.
English descriptors
- KwdEn :
- Animals, Chlorocebus aethiops, Coronavirus, DNA Primers (chemistry), Detection, Method, Microbiology, Nucleic Acid Amplification Techniques (instrumentation), Polymerase chain reaction, RNA, Viral (analysis), Reagent Kits, Diagnostic, Real time, Reverse transcription polymerase chain reaction, SARS Virus (genetics), SARS Virus (isolation & purification), Sensitivity, Sensitivity and Specificity, Severe Acute Respiratory Syndrome (virology), Severe acute respiratory syndrome, Specificity, Vero Cells.
- MESH :
- chemical , analysis : RNA, Viral.
- chemical , chemistry : DNA Primers.
- genetics : SARS Virus.
- instrumentation : Nucleic Acid Amplification Techniques.
- isolation & purification : SARS Virus.
- virology : Severe Acute Respiratory Syndrome.
- Animals, Chlorocebus aethiops, Reagent Kits, Diagnostic, Sensitivity and Specificity, Vero Cells.
Abstract
The sensitivity and specificity of various severe acute respiratory syndrome coronavirus (SARS-CoV) PCR primer and probe sets were evaluated through the use of commercial kits and in-house amplification formats. Conventional and real-time PCR assays were performed using a heat-block thermocycler ABI 9600, the Roche LightCycler version 1.2, or the ABI 7000 Sequence Detection System. The sensitivity of all primers was between 0.0004 and 0.04 PFU with viral cell lysate and between 0.004 and 0.4 PFU in spiked stool specimen per PCR assay. The primer sets for real-time PCR assays were at one least 1 log more sensitive than the primer sets used in the conventional PCR. A panel of viruses including swine gastroenteritis virus, bovine coronavirus, avian bronchitis virus (Connecticut strain), avian bronchitis virus (Massachusetts strain), human coronaviruses 229E and OC43, parainfluenza virus (type III), human metapneumovirus, adenovirus, respiratory syncytial virus, and influenza A were tested by all assays. All real-time PCR assays used probe-based detection, and no cross-reactivity was observed. With conventional PCR, analysis was performed using agarose gel electrophoresis and multiple nonspecific bands were observed. Two commercial extraction methods, magnetic particle capture and silica-based procedure were evaluated and the results were comparable. The former was less laborious with shorter time for completion and can easily be adapted to an automated system such as the MagNa Pure-LC, which can extract nucleic acid from clinical samples and load it into the sample capillaries of the LightCycler. As exemplified by this study, the continued refinement and evaluation of PCR procedures will greatly benefit the diagnostic laboratory during an outbreak of SARS.
Url:
Affiliations:
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Le document en format XML
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Michael" sort="Drebot, Michael" uniqKey="Drebot M" first="Michael" last="Drebot">Michael Drebot</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>National Microbiology Laboratory</s1><s2>Winnipeg, Manitoba, R3E 2R2</s2><s3>CAN</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>Canada</country><wicri:noRegion>National Microbiology Laboratory</wicri:noRegion></affiliation></author><author><name sortKey="Andonov, Anton" sort="Andonov, Anton" uniqKey="Andonov A" first="Anton" last="Andonov">Anton Andonov</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>National Microbiology Laboratory</s1><s2>Winnipeg, Manitoba, R3E 2R2</s2><s3>CAN</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ></inist:fA14><country>Canada</country><wicri:noRegion>National Microbiology Laboratory</wicri:noRegion></affiliation></author><author><name sortKey="Petrich, Astrid" sort="Petrich, Astrid" uniqKey="Petrich A" first="Astrid" last="Petrich">Astrid Petrich</name><affiliation wicri:level="4"><inist:fA14 i1="03"><s1>St. Joseph's Healthcare, McMaster University</s1><s2>Hamilton Ontario, L8N 4A6</s2><s3>CAN</s3><sZ>4 aut.</sZ><sZ>6 aut.</sZ></inist:fA14><country>Canada</country><placeName><settlement type="city">Hamilton (Ontario)</settlement><region type="state">Ontario</region></placeName><orgName type="university">Université McMaster</orgName></affiliation></author><author><name sortKey="Glushek, Martin" sort="Glushek, Martin" uniqKey="Glushek M" first="Martin" last="Glushek">Martin Glushek</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Provincial Laboratory for Public Health (Microbiology), University of Alberta Hospital</s1><s2>Edmonton, Alberta, T6G 2J2</s2><s3>CAN</s3><sZ>1 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>Canada</country><wicri:noRegion>Edmonton, Alberta, T6G 2J2</wicri:noRegion></affiliation></author><author><name sortKey="Mahony, James" sort="Mahony, James" uniqKey="Mahony J" first="James" last="Mahony">James Mahony</name><affiliation wicri:level="4"><inist:fA14 i1="03"><s1>St. Joseph's Healthcare, McMaster University</s1><s2>Hamilton Ontario, L8N 4A6</s2><s3>CAN</s3><sZ>4 aut.</sZ><sZ>6 aut.</sZ></inist:fA14><country>Canada</country><placeName><settlement type="city">Hamilton (Ontario)</settlement><region type="state">Ontario</region></placeName><orgName type="university">Université McMaster</orgName></affiliation></author></analytic><series><title level="j" type="main">Diagnostic microbiology and infectious disease</title><title level="j" type="abbreviated">Diagn. microbiol. infect. dis.</title><idno type="ISSN">0732-8893</idno><imprint><date when="2005">2005</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Diagnostic microbiology and infectious disease</title><title level="j" type="abbreviated">Diagn. microbiol. infect. dis.</title><idno type="ISSN">0732-8893</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Chlorocebus aethiops</term><term>Coronavirus</term><term>DNA Primers (chemistry)</term><term>Detection</term><term>Method</term><term>Microbiology</term><term>Nucleic Acid Amplification Techniques (instrumentation)</term><term>Polymerase chain reaction</term><term>RNA, Viral (analysis)</term><term>Reagent Kits, Diagnostic</term><term>Real time</term><term>Reverse transcription polymerase chain reaction</term><term>SARS Virus (genetics)</term><term>SARS Virus (isolation & purification)</term><term>Sensitivity</term><term>Sensitivity and Specificity</term><term>Severe Acute Respiratory Syndrome (virology)</term><term>Severe acute respiratory syndrome</term><term>Specificity</term><term>Vero Cells</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN viral (analyse)</term><term>Amorces ADN ()</term><term>Animaux</term><term>Cellules Vero</term><term>Sensibilité et spécificité</term><term>Syndrome respiratoire aigu sévère (virologie)</term><term>Techniques d'amplification d'acides nucléiques (instrumentation)</term><term>Trousses de réactifs pour diagnostic</term><term>Virus du SRAS (génétique)</term><term>Virus du SRAS (isolement et purification)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>RNA, Viral</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>DNA Primers</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>ARN viral</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Virus du SRAS</term></keywords><keywords scheme="MESH" qualifier="instrumentation" xml:lang="en"><term>Nucleic Acid Amplification Techniques</term></keywords><keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Virus du SRAS</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>Animals</term><term>Chlorocebus aethiops</term><term>Reagent Kits, Diagnostic</term><term>Sensitivity and Specificity</term><term>Vero Cells</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Amorces ADN</term><term>Animaux</term><term>Cellules Vero</term><term>Coronavirus</term><term>Réaction chaîne polymérase</term><term>Détection</term><term>Méthode</term><term>Sensibilité et spécificité</term><term>Techniques d'amplification d'acides nucléiques</term><term>Temps réel</term><term>Réaction chaîne polymérase RT</term><term>Sensibilité</term><term>Spécificité</term><term>Microbiologie</term><term>Syndrome respiratoire aigu sévère</term><term>Trousses de réactifs pour diagnostic</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The sensitivity and specificity of various severe acute respiratory syndrome coronavirus (SARS-CoV) PCR primer and probe sets were evaluated through the use of commercial kits and in-house amplification formats. Conventional and real-time PCR assays were performed using a heat-block thermocycler ABI 9600, the Roche LightCycler version 1.2, or the ABI 7000 Sequence Detection System. The sensitivity of all primers was between 0.0004 and 0.04 PFU with viral cell lysate and between 0.004 and 0.4 PFU in spiked stool specimen per PCR assay. The primer sets for real-time PCR assays were at one least 1 log more sensitive than the primer sets used in the conventional PCR. A panel of viruses including swine gastroenteritis virus, bovine coronavirus, avian bronchitis virus (Connecticut strain), avian bronchitis virus (Massachusetts strain), human coronaviruses 229E and OC43, parainfluenza virus (type III), human metapneumovirus, adenovirus, respiratory syncytial virus, and influenza A were tested by all assays. All real-time PCR assays used probe-based detection, and no cross-reactivity was observed. With conventional PCR, analysis was performed using agarose gel electrophoresis and multiple nonspecific bands were observed. Two commercial extraction methods, magnetic particle capture and silica-based procedure were evaluated and the results were comparable. The former was less laborious with shorter time for completion and can easily be adapted to an automated system such as the MagNa Pure-LC, which can extract nucleic acid from clinical samples and load it into the sample capillaries of the LightCycler. As exemplified by this study, the continued refinement and evaluation of PCR procedures will greatly benefit the diagnostic laboratory during an outbreak of SARS.</div></front></TEI><affiliations><list><country><li>Canada</li></country><region><li>Ontario</li></region><settlement><li>Hamilton (Ontario)</li></settlement><orgName><li>Université McMaster</li></orgName></list><tree><country name="Canada"><noRegion><name sortKey="Chui, Linda" sort="Chui, Linda" uniqKey="Chui L" first="Linda" last="Chui">Linda Chui</name></noRegion><name sortKey="Andonov, Anton" sort="Andonov, Anton" uniqKey="Andonov A" first="Anton" last="Andonov">Anton Andonov</name><name sortKey="Drebot, Michael" sort="Drebot, Michael" uniqKey="Drebot M" first="Michael" last="Drebot">Michael Drebot</name><name sortKey="Glushek, Martin" sort="Glushek, Martin" uniqKey="Glushek M" first="Martin" last="Glushek">Martin Glushek</name><name sortKey="Mahony, James" sort="Mahony, James" uniqKey="Mahony J" first="James" last="Mahony">James Mahony</name><name sortKey="Petrich, Astrid" sort="Petrich, Astrid" uniqKey="Petrich A" first="Astrid" last="Petrich">Astrid Petrich</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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