Design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona- and toroviruses
Identifieur interne : 000D00 ( Main/Exploration ); précédent : 000C99; suivant : 000D01Design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona- and toroviruses
Auteurs : Kalina T. Zlateva [Pays-Bas] ; Kelly M. Crusio [Pays-Bas] ; Andrey M. Leontovich [Russie] ; Chris Lauber [Pays-Bas] ; Eric Claas [Pays-Bas] ; Alexander A. Kravchenko [Russie] ; Willy J. M. Spaan [Pays-Bas] ; Alexander E. Gorbalenya [Pays-Bas, Russie]Source :
- Journal of virological methods [ 0166-0934 ] ; 2011.
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- Pascal (Inist)
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Abstract
The ssRNA+ family Coronaviridae includes two subfamilies prototyped by coronaviruses and toroviruses that cause respiratory and enteric infections. To facilitate the identification of new distantly related members of the family Coronaviridae, we have developed a molecular assay with broad specificity. The consensus-degenerated hybrid oligonucleotide primer (CODEHOP) strategy was modified to design primers targeting the most conserved motifs in the RNA-dependent RNA polymerase locus. They were evaluated initially on RNA templates from virus-infected cells using a two-step RT-PCR protocol that was further advanced to a one-step assay. The sensitivity of the assay ranged from 102 to 106 and from 105 to 109 RNA copy numbers for individual corona-/torovirus templates when tested, respectively, with and without an excess of RNA from human cells. This primer set compared to that designed according to the original CODEHOP rules showed 10-103 folds greater sensitivity for 5 of the 6 evaluated corona-/torovirus templates. It detected 57% (32 of 56) of the respiratory specimens positive for 4 human coronaviruses, as well as stool specimens positive for a bovine torovirus. The high sensitivity and broad virus range of this assay makes it suitable for screening biological specimens in search for new viruses of the family Coronaviridae.
Affiliations:
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Zlateva</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Department of Medical Microbiology, Leiden University Medical Center</s1><s2>2300 RC Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>Pays-Bas</country><wicri:noRegion>2300 RC Leiden</wicri:noRegion></affiliation></author><author><name sortKey="Crusio, Kelly M" sort="Crusio, Kelly M" uniqKey="Crusio K" first="Kelly M." last="Crusio">Kelly M. Crusio</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Department of Medical Microbiology, Leiden University Medical Center</s1><s2>2300 RC Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>Pays-Bas</country><wicri:noRegion>2300 RC Leiden</wicri:noRegion></affiliation></author><author><name sortKey="Leontovich, Andrey M" sort="Leontovich, Andrey M" uniqKey="Leontovich A" first="Andrey M." last="Leontovich">Andrey M. Leontovich</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>The AN. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University</s1><s2>119899 Moscow</s2><s3>RUS</s3><sZ>3 aut.</sZ><sZ>6 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>Russie</country><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation></author><author><name sortKey="Lauber, Chris" sort="Lauber, Chris" uniqKey="Lauber C" first="Chris" last="Lauber">Chris Lauber</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Department of Medical Microbiology, Leiden University Medical Center</s1><s2>2300 RC Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>Pays-Bas</country><wicri:noRegion>2300 RC Leiden</wicri:noRegion></affiliation></author><author><name sortKey="Claas, Eric" sort="Claas, Eric" uniqKey="Claas E" first="Eric" last="Claas">Eric Claas</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Department of Medical Microbiology, Leiden University Medical Center</s1><s2>2300 RC Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>Pays-Bas</country><wicri:noRegion>2300 RC Leiden</wicri:noRegion></affiliation></author><author><name sortKey="Kravchenko, Alexander A" sort="Kravchenko, Alexander A" uniqKey="Kravchenko A" first="Alexander A." last="Kravchenko">Alexander A. Kravchenko</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>The AN. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University</s1><s2>119899 Moscow</s2><s3>RUS</s3><sZ>3 aut.</sZ><sZ>6 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>Russie</country><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation></author><author><name sortKey="Spaan, Willy J M" sort="Spaan, Willy J M" uniqKey="Spaan W" first="Willy J. M." last="Spaan">Willy J. M. Spaan</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Department of Medical Microbiology, Leiden University Medical Center</s1><s2>2300 RC Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>Pays-Bas</country><wicri:noRegion>2300 RC Leiden</wicri:noRegion></affiliation></author><author><name sortKey="Gorbalenya, Alexander E" sort="Gorbalenya, Alexander E" uniqKey="Gorbalenya A" first="Alexander E." last="Gorbalenya">Alexander E. Gorbalenya</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Department of Medical Microbiology, Leiden University Medical Center</s1><s2>2300 RC Leiden</s2><s3>NLD</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>Pays-Bas</country><wicri:noRegion>2300 RC Leiden</wicri:noRegion></affiliation><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>The AN. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University</s1><s2>119899 Moscow</s2><s3>RUS</s3><sZ>3 aut.</sZ><sZ>6 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>Russie</country><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation><affiliation wicri:level="1"><inist:fA14 i1="03"><s1>Faculty of Bioengineering and Bioinformatics, M.V. Lomonosov Moscow State University</s1><s2>119899 Moscow</s2><s3>RUS</s3><sZ>8 aut.</sZ></inist:fA14><country>Russie</country><placeName><settlement type="city">Moscou</settlement><region>District fédéral central</region></placeName></affiliation></author></analytic><series><title level="j" type="main">Journal of virological methods</title><title level="j" type="abbreviated">J. virol. methods</title><idno type="ISSN">0166-0934</idno><imprint><date when="2011">2011</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Journal of virological methods</title><title level="j" type="abbreviated">J. virol. methods</title><idno type="ISSN">0166-0934</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Coronavirus</term><term>Detection</term><term>Method</term><term>Polymerase chain reaction</term><term>RNA-directed RNA polymerase</term><term>Torovirus</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Coronavirus</term><term>Torovirus</term><term>Détection</term><term>RNA-directed RNA polymerase</term><term>Réaction chaîne polymérase</term><term>Méthode</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The ssRNA+ family Coronaviridae includes two subfamilies prototyped by coronaviruses and toroviruses that cause respiratory and enteric infections. To facilitate the identification of new distantly related members of the family Coronaviridae, we have developed a molecular assay with broad specificity. The consensus-degenerated hybrid oligonucleotide primer (CODEHOP) strategy was modified to design primers targeting the most conserved motifs in the RNA-dependent RNA polymerase locus. They were evaluated initially on RNA templates from virus-infected cells using a two-step RT-PCR protocol that was further advanced to a one-step assay. The sensitivity of the assay ranged from 10<sup>2</sup> to 10<sup>6</sup> and from 10<sup>5</sup> to 10<sup>9</sup> RNA copy numbers for individual corona-/torovirus templates when tested, respectively, with and without an excess of RNA from human cells. This primer set compared to that designed according to the original CODEHOP rules showed 10-103 folds greater sensitivity for 5 of the 6 evaluated corona-/torovirus templates. It detected 57% (32 of 56) of the respiratory specimens positive for 4 human coronaviruses, as well as stool specimens positive for a bovine torovirus. The high sensitivity and broad virus range of this assay makes it suitable for screening biological specimens in search for new viruses of the family Coronaviridae.</div></front></TEI><affiliations><list><country><li>Pays-Bas</li><li>Russie</li></country><region><li>District fédéral central</li></region><settlement><li>Moscou</li></settlement></list><tree><country name="Pays-Bas"><noRegion><name sortKey="Zlateva, Kalina T" sort="Zlateva, Kalina T" uniqKey="Zlateva K" first="Kalina T." last="Zlateva">Kalina T. Zlateva</name></noRegion><name sortKey="Claas, Eric" sort="Claas, Eric" uniqKey="Claas E" first="Eric" last="Claas">Eric Claas</name><name sortKey="Crusio, Kelly M" sort="Crusio, Kelly M" uniqKey="Crusio K" first="Kelly M." last="Crusio">Kelly M. Crusio</name><name sortKey="Gorbalenya, Alexander E" sort="Gorbalenya, Alexander E" uniqKey="Gorbalenya A" first="Alexander E." last="Gorbalenya">Alexander E. Gorbalenya</name><name sortKey="Lauber, Chris" sort="Lauber, Chris" uniqKey="Lauber C" first="Chris" last="Lauber">Chris Lauber</name><name sortKey="Spaan, Willy J M" sort="Spaan, Willy J M" uniqKey="Spaan W" first="Willy J. M." last="Spaan">Willy J. M. Spaan</name></country><country name="Russie"><region name="District fédéral central"><name sortKey="Leontovich, Andrey M" sort="Leontovich, Andrey M" uniqKey="Leontovich A" first="Andrey M." last="Leontovich">Andrey M. Leontovich</name></region><name sortKey="Gorbalenya, Alexander E" sort="Gorbalenya, Alexander E" uniqKey="Gorbalenya A" first="Alexander E." last="Gorbalenya">Alexander E. Gorbalenya</name><name sortKey="Gorbalenya, Alexander E" sort="Gorbalenya, Alexander E" uniqKey="Gorbalenya A" first="Alexander E." last="Gorbalenya">Alexander E. Gorbalenya</name><name sortKey="Kravchenko, Alexander A" sort="Kravchenko, Alexander A" uniqKey="Kravchenko A" first="Alexander A." last="Kravchenko">Alexander A. Kravchenko</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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