Development of Taqman RT-nested PCR system for clinical SARS-CoV detection.
Severe acute respiratory syndrome (SARS) is an acute newly emerged infectious respiratory illness. The etiologic agent of SARS was named 'SARS-associated coronavirus' (SARS-CoV) that can be detected with reverse transcription-polymerase chain reaction (RT-PCR) assays. In this study, 12 sets of nested primers covering the SARS-CoV genome have been screened and showed sufficient sensitivity to detect SARS-CoV in RNA isolated from virus cultured in Vero 6 cells. To optimize further the reaction condition of those nested primers sets, seven sets of nested primers have been chosen to compare their reverse transcribed efficiency with specific and random primers, which is useful to combine RT with the first round of PCR into a one-step RT-PCR. Based on the sensitivity and simplicity of results, the no. 73 primer set was chosen as the candidate primer set for clinical diagnoses. To specify the amplicon to minimize false positive results, a Taqman RT-nested PCR system of no. 73 nested primer set was developed. Through investigations on a test panel of whole blood obtained from 30 SARS patients and 9 control persons, the specificity and sensitivity of the Taqman RT-nested PCR system was found to be 100 and 83%, respectively, which suggests that the method is a promising one to diagnose SARS in early stages.
Wu
Qingfa
Q
Beijing Genomics Institute, Chinese Academy of Sciences, Airport Industrial Zone, Bldg 6, Beijing 101300, PR China.
Xu
Zuyuan
Z
Wei
Tian
T
Zeng
Haipang
H
Li
Jingxiang
J
Gang
Haixue
H
Sun
Min
M
Jiang
Fangbo
F
Wang
Xiang
X
Dong
Wei
W
Yang
Ling
L
Wang
Jian
J
D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D002648:Child; D002681:China; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D016679:Genome, Viral; D006801:Humans; D008297:Male; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology*; D014773:Virology / Q000379:methods / Q000706:statistics & numerical data
D017931:DNA Primers; D004279:DNA, Viral