Comparison of 9 different PCR primers for the rapid detection of severe acute respiratory syndrome coronavirus using 2 RNA extraction methods.
Identifieur interne : 002786 ( PubMed/Checkpoint ); précédent : 002785; suivant : 002787Comparison 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 ; Anton Andonov ; Astrid Petrich ; Martin Glushek ; James MahonySource :
- 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.
- Amorces ADN, Animaux, Cellules Vero, Sensibilité et spécificité, Techniques d'amplification d'acides nucléiques, Trousses de réactifs pour diagnostic.
English descriptors
- KwdEn :
- Animals, Chlorocebus aethiops, DNA Primers (chemistry), Nucleic Acid Amplification Techniques (instrumentation), RNA, Viral (analysis), Reagent Kits, Diagnostic, SARS Virus (genetics), SARS Virus (isolation & purification), Sensitivity and Specificity, Severe Acute Respiratory Syndrome (virology), 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.
DOI: 10.1016/j.diagmicrobio.2005.03.007
PubMed: 15994050
Affiliations:
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first="Anton" last="Andonov">Anton Andonov</name></author><author><name sortKey="Petrich, Astrid" sort="Petrich, Astrid" uniqKey="Petrich A" first="Astrid" last="Petrich">Astrid Petrich</name></author><author><name sortKey="Glushek, Martin" sort="Glushek, Martin" uniqKey="Glushek M" first="Martin" last="Glushek">Martin Glushek</name></author><author><name sortKey="Mahony, James" sort="Mahony, James" uniqKey="Mahony J" first="James" last="Mahony">James Mahony</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2005">2005</date><idno type="RBID">pubmed:15994050</idno><idno type="pmid">15994050</idno><idno type="doi">10.1016/j.diagmicrobio.2005.03.007</idno><idno type="wicri:Area/PubMed/Corpus">002658</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002658</idno><idno type="wicri:Area/PubMed/Curation">002658</idno><idno type="wicri:explorRef" wicri:stream="PubMed" 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sort="Drebot, Michael" uniqKey="Drebot M" first="Michael" last="Drebot">Michael Drebot</name></author><author><name sortKey="Andonov, Anton" sort="Andonov, Anton" uniqKey="Andonov A" first="Anton" last="Andonov">Anton Andonov</name></author><author><name sortKey="Petrich, Astrid" sort="Petrich, Astrid" uniqKey="Petrich A" first="Astrid" last="Petrich">Astrid Petrich</name></author><author><name sortKey="Glushek, Martin" sort="Glushek, Martin" uniqKey="Glushek M" first="Martin" last="Glushek">Martin Glushek</name></author><author><name sortKey="Mahony, James" sort="Mahony, James" uniqKey="Mahony J" first="James" last="Mahony">James Mahony</name></author></analytic><series><title level="j">Diagnostic microbiology and infectious disease</title><idno type="ISSN">0732-8893</idno><imprint><date when="2005" type="published">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Chlorocebus aethiops</term><term>DNA Primers (chemistry)</term><term>Nucleic Acid Amplification Techniques (instrumentation)</term><term>RNA, Viral (analysis)</term><term>Reagent Kits, Diagnostic</term><term>SARS Virus (genetics)</term><term>SARS Virus (isolation & purification)</term><term>Sensitivity and Specificity</term><term>Severe Acute Respiratory Syndrome (virology)</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="MESH" xml:lang="fr"><term>Amorces ADN</term><term>Animaux</term><term>Cellules Vero</term><term>Sensibilité et spécificité</term><term>Techniques d'amplification d'acides nucléiques</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><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">15994050</PMID><DateCompleted><Year>2006</Year><Month>02</Month><Day>16</Day></DateCompleted><DateRevised><Year>2020</Year><Month>04</Month><Day>09</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0732-8893</ISSN><JournalIssue CitedMedium="Print"><Volume>53</Volume><Issue>1</Issue><PubDate><Year>2005</Year><Month>Sep</Month></PubDate></JournalIssue><Title>Diagnostic microbiology and infectious disease</Title><ISOAbbreviation>Diagn. Microbiol. Infect. Dis.</ISOAbbreviation></Journal><ArticleTitle>Comparison of 9 different PCR primers for the rapid detection of severe acute respiratory syndrome coronavirus using 2 RNA extraction methods.</ArticleTitle><Pagination><MedlinePgn>47-55</MedlinePgn></Pagination><Abstract><AbstractText>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.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Chui</LastName><ForeName>Linda</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>Provincial Laboratory for Public Health (Microbiology), University of Alberta Hospital, Edmonton, Alberta, Canada T6G 2J2. lchui@provlab.ab.ca</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Drebot</LastName><ForeName>Michael</ForeName><Initials>M</Initials></Author><Author ValidYN="Y"><LastName>Andonov</LastName><ForeName>Anton</ForeName><Initials>A</Initials></Author><Author ValidYN="Y"><LastName>Petrich</LastName><ForeName>Astrid</ForeName><Initials>A</Initials></Author><Author ValidYN="Y"><LastName>Glushek</LastName><ForeName>Martin</ForeName><Initials>M</Initials></Author><Author ValidYN="Y"><LastName>Mahony</LastName><ForeName>James</ForeName><Initials>J</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D003160">Comparative Study</PublicationType><PublicationType UI="D023362">Evaluation Study</PublicationType><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Diagn Microbiol Infect Dis</MedlineTA><NlmUniqueID>8305899</NlmUniqueID><ISSNLinking>0732-8893</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D017931">DNA Primers</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D012367">RNA, Viral</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011933">Reagent Kits, 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IdType="pubmed">14522060</ArticleId></ArticleIdList></Reference></ReferenceList></PubmedData></pubmed><affiliations><list><country><li>Canada</li></country></list><tree><noCountry><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></noCountry><country name="Canada"><noRegion><name sortKey="Chui, Linda" sort="Chui, Linda" uniqKey="Chui L" first="Linda" last="Chui">Linda 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