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Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens

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Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens

Auteurs : S C C. Wong [République populaire de Chine] ; J K C. Chan [République populaire de Chine] ; K C Lee [République populaire de Chine] ; E S F. Lo [République populaire de Chine] ; D N C. Tsang [République populaire de Chine]

Source :

Journal of Clinical Pathology [ 0021-9746 ] ; 2005-03.

RBID : ISTEX:AE7CAE9B9EA1F3FDB3F1B19A0CD0D80DD8BA42AE

English descriptors

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 χ2 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 (χ2 = 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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https://api.istex.fr/ark:/67375/NVC-CNFP4K1H-1/fulltext.pdf

DOI: 10.1136/jcp.2004.016592

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<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>
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<term>RT-PCR, reverse transcriptase polymerase chain reaction</term>
<term>SARS, severe acute respiratory syndrome</term>
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<term>Healthy controls</term>
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<term>Primer</term>
<term>Queen elizabeth hospital</term>
<term>Rectal</term>
<term>Respiratory syndrome</term>
<term>Respiratory syndrome coronavirus</term>
<term>Sampling techniques</term>
<term>Sars</term>
<term>Sars coronavirus</term>
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<term>Significant difference</term>
<term>Statistical analysis</term>
<term>Stool samples</term>
<term>Swab</term>
<term>Syndrome</term>
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<term>Transcriptase</term>
<term>Transcriptase polymerase chain reaction</term>
<term>Transcriptase polymerase chain reaction assay</term>
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<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 χ2 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 (χ2 = 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>
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Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens

Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens

Source :

English descriptors

Abstract

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