Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens
Identifieur interne : 000282 ( PascalFrancis/Curation ); précédent : 000281; suivant : 000283Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens
Auteurs : S. C. C. Wong [Hong Kong] ; J. K. C. Chan [Hong Kong] ; K. C. Lee [Hong Kong] ; E. S. F. Lo [Hong Kong] ; D. N. C. Tsang [Hong Kong]Source :
- Journal of clinical pathology [ 0021-9746 ] ; 2005.
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- Pascal (Inist)
- Wicri :
- topic : Analyse quantitative.
English descriptors
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Abstract
Aims: To develop a quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) for severe acute respiratory syndrome coronavirus (SARS-CoV) detection and explore the potential of using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control to exclude false negative results. Methods: SARS-CoV and GAPDH mRNA were both measured in 26 specimens from 16 patients with SARS, 40 follow up specimens from the same batch of patients, and appropriate control subjects. The relation between SARS positivity and GAPDH mRNA concentration was investigated using the X2 test. Increasing the sensitivity for SARS-CoV and GAPDH mRNA detection was investigated in follow up specimens in which SARS-CoV and GAPDH mRNA were not detected initially. Results: Varying amounts of SARS-CoV were found in the 26 SARS-CoV positive specimens and SARS-CoV was not detected in the 40 follow up specimens and controls. In addition, concentrations of GAPDH mRNA were significantly different between the patients with SARS, follow up specimens, and healthy controls (Kruskal-Wallis test, p<0.05). Moreover, GAPDH mRNA concentrations were highly correlated with SARS-CoV positivity (X2 = 5.43; p<0.05). Finally, SARS-CoV and GAPDH mRNA were both detected in three follow up urine specimens that were initially negative when the amount of cDNA used was increased from 5 μl to 10 and 15 μl. Conclusions: This Q-RT-PCR assay can be used to detect SARS-CoV. Moreover, GAPDH mRNA may be useful to rule out false negative results in SARS-CoV detection, and the current extraction method for urine may not be sensitive enough to detect low titres of SARS-CoV.
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en" level="a">Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens</title><author><name sortKey="Wong, S C C" sort="Wong, S C C" uniqKey="Wong S" first="S. C. C." last="Wong">S. C. C. Wong</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Department of Clinical Oncology, Prince of Wales Hospital, The Chinese University of Hong Kong</s1><s3>HKG</s3><sZ>1 aut.</sZ></inist:fA14><country>Hong Kong</country></affiliation></author><author><name sortKey="Chan, J K C" sort="Chan, J K C" uniqKey="Chan J" first="J. K. C." last="Chan">J. K. C. Chan</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Department of Pathology, Queen Elizabeth Hospital</s1><s3>HKG</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>Hong Kong</country></affiliation></author><author><name sortKey="Lee, K C" sort="Lee, K C" uniqKey="Lee K" first="K. C." last="Lee">K. C. Lee</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Department of Pathology, Queen Elizabeth Hospital</s1><s3>HKG</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>Hong Kong</country></affiliation></author><author><name sortKey="Lo, E S F" sort="Lo, E S F" uniqKey="Lo E" first="E. S. F." last="Lo">E. S. F. Lo</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Department of Pathology, Queen Elizabeth Hospital</s1><s3>HKG</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>Hong Kong</country></affiliation></author><author><name sortKey="Tsang, D N C" sort="Tsang, D N C" uniqKey="Tsang D" first="D. N. C." last="Tsang">D. N. C. 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Wong</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Department of Clinical Oncology, Prince of Wales Hospital, The Chinese University of Hong Kong</s1><s3>HKG</s3><sZ>1 aut.</sZ></inist:fA14><country>Hong Kong</country></affiliation></author><author><name sortKey="Chan, J K C" sort="Chan, J K C" uniqKey="Chan J" first="J. K. C." last="Chan">J. K. C. Chan</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Department of Pathology, Queen Elizabeth Hospital</s1><s3>HKG</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>Hong Kong</country></affiliation></author><author><name sortKey="Lee, K C" sort="Lee, K C" uniqKey="Lee K" first="K. C." last="Lee">K. C. Lee</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Department of Pathology, Queen Elizabeth Hospital</s1><s3>HKG</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>Hong Kong</country></affiliation></author><author><name sortKey="Lo, E S F" sort="Lo, E S F" uniqKey="Lo E" first="E. S. F." last="Lo">E. S. F. Lo</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Department of Pathology, Queen Elizabeth Hospital</s1><s3>HKG</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>Hong Kong</country></affiliation></author><author><name sortKey="Tsang, D N C" sort="Tsang, D N C" uniqKey="Tsang D" first="D. N. C." last="Tsang">D. N. C. Tsang</name><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Department of Pathology, Queen Elizabeth Hospital</s1><s3>HKG</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></inist:fA14><country>Hong Kong</country></affiliation></author></analytic><series><title level="j" type="main">Journal of clinical pathology</title><title level="j" type="abbreviated">J. clin. pathol.</title><idno type="ISSN">0021-9746</idno><imprint><date when="2005">2005</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Journal of clinical pathology</title><title level="j" type="abbreviated">J. clin. pathol.</title><idno type="ISSN">0021-9746</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Anatomic pathology</term><term>Coronavirus</term><term>Correlation</term><term>Development</term><term>Messenger RNA</term><term>Quantitative analysis</term><term>Severe acute respiratory syndrome</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Développement</term><term>Analyse quantitative</term><term>Syndrome respiratoire aigu sévère</term><term>Coronavirus</term><term>Corrélation</term><term>RNA messager</term><term>Anatomopathologie</term></keywords><keywords scheme="Wicri" type="topic" xml:lang="fr"><term>Analyse quantitative</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Aims: To develop a quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) for severe acute respiratory syndrome coronavirus (SARS-CoV) detection and explore the potential of using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control to exclude false negative results. Methods: SARS-CoV and GAPDH mRNA were both measured in 26 specimens from 16 patients with SARS, 40 follow up specimens from the same batch of patients, and appropriate control subjects. The relation between SARS positivity and GAPDH mRNA concentration was investigated using the X<sup>2</sup> test. Increasing the sensitivity for SARS-CoV and GAPDH mRNA detection was investigated in follow up specimens in which SARS-CoV and GAPDH mRNA were not detected initially. Results: Varying amounts of SARS-CoV were found in the 26 SARS-CoV positive specimens and SARS-CoV was not detected in the 40 follow up specimens and controls. In addition, concentrations of GAPDH mRNA were significantly different between the patients with SARS, follow up specimens, and healthy controls (Kruskal-Wallis test, p<0.05). Moreover, GAPDH mRNA concentrations were highly correlated with SARS-CoV positivity (X<sup>2</sup> = 5.43; p<0.05). Finally, SARS-CoV and GAPDH mRNA were both detected in three follow up urine specimens that were initially negative when the amount of cDNA used was increased from 5 μl to 10 and 15 μl. Conclusions: This Q-RT-PCR assay can be used to detect SARS-CoV. Moreover, GAPDH mRNA may be useful to rule out false negative results in SARS-CoV detection, and the current extraction method for urine may not be sensitive enough to detect low titres of SARS-CoV.</div></front></TEI><inist><standard h6="B"><pA><fA01 i1="01" i2="1"><s0>0021-9746</s0></fA01><fA02 i1="01"><s0>JCPAAK</s0></fA02><fA03 i2="1"><s0>J. clin. pathol.</s0></fA03><fA05><s2>58</s2></fA05><fA06><s2>3</s2></fA06><fA08 i1="01" i2="1" l="ENG"><s1>Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens</s1></fA08><fA11 i1="01" i2="1"><s1>WONG (S. C. C.)</s1></fA11><fA11 i1="02" i2="1"><s1>CHAN (J. K. C.)</s1></fA11><fA11 i1="03" i2="1"><s1>LEE (K. C.)</s1></fA11><fA11 i1="04" i2="1"><s1>LO (E. S. F.)</s1></fA11><fA11 i1="05" i2="1"><s1>TSANG (D. N. C.)</s1></fA11><fA14 i1="01"><s1>Department of Clinical Oncology, Prince of Wales Hospital, The Chinese University of Hong Kong</s1><s3>HKG</s3><sZ>1 aut.</sZ></fA14><fA14 i1="02"><s1>Department of Pathology, Queen Elizabeth Hospital</s1><s3>HKG</s3><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ></fA14><fA20><s1>276-280</s1></fA20><fA21><s1>2005</s1></fA21><fA23 i1="01"><s0>ENG</s0></fA23><fA43 i1="01"><s1>INIST</s1><s2>3020</s2><s5>354000126908070100</s5></fA43><fA44><s0>0000</s0><s1>© 2005 INIST-CNRS. All rights reserved.</s1></fA44><fA45><s0>21 ref.</s0></fA45><fA47 i1="01" i2="1"><s0>05-0168771</s0></fA47><fA60><s1>P</s1></fA60><fA61><s0>A</s0></fA61><fA64 i1="01" i2="1"><s0>Journal of clinical pathology</s0></fA64><fA66 i1="01"><s0>GBR</s0></fA66><fC01 i1="01" l="ENG"><s0>Aims: To develop a quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) for severe acute respiratory syndrome coronavirus (SARS-CoV) detection and explore the potential of using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control to exclude false negative results. Methods: SARS-CoV and GAPDH mRNA were both measured in 26 specimens from 16 patients with SARS, 40 follow up specimens from the same batch of patients, and appropriate control subjects. The relation between SARS positivity and GAPDH mRNA concentration was investigated using the X<sup>2</sup> test. Increasing the sensitivity for SARS-CoV and GAPDH mRNA detection was investigated in follow up specimens in which SARS-CoV and GAPDH mRNA were not detected initially. Results: Varying amounts of SARS-CoV were found in the 26 SARS-CoV positive specimens and SARS-CoV was not detected in the 40 follow up specimens and controls. In addition, concentrations of GAPDH mRNA were significantly different between the patients with SARS, follow up specimens, and healthy controls (Kruskal-Wallis test, p<0.05). Moreover, GAPDH mRNA concentrations were highly correlated with SARS-CoV positivity (X<sup>2</sup> = 5.43; p<0.05). Finally, SARS-CoV and GAPDH mRNA were both detected in three follow up urine specimens that were initially negative when the amount of cDNA used was increased from 5 μl to 10 and 15 μl. Conclusions: This Q-RT-PCR assay can be used to detect SARS-CoV. 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