Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens.
OBJECTIVE
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 chi2 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 (chi2 = 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 microl to 10 and 15 microl.


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.
Wong
S C C
SC
Department of Clinical Oncology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China. cesar@clo.cuhk.edu.hk
Chan
J K C
JK
Lee
K C
KC
Lo
E S F
ES
Tsang
D N C
DN
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000378:metabolism; D005188:False Negative Reactions; D005260:Female; D005500:Follow-Up Studies; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D011786:Quality Control; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D045473:Severe acute respiratory syndrome-related coronavirus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis*
D000199:Actins; D015415:Biomarkers; D012333:RNA, Messenger; D012367:RNA, Viral; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
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