Sensitive detection of SARS coronavirus RNA by a novel asymmetric multiplex nested RT-PCR amplification coupled with oligonucleotide microarray hybridization.
We have developed a sensitive method for the detection of specific genes simultaneously. First, DNA was amplified by a novel asymmetric multiplex PCR with universal primer(s). Second, the 6-carboxytetramethylrhodamine (TAMRA)-labeled PCR products were hybridized specifically with oligonucleotide microarrays. Finally, matched duplexes were detected by using a laser-induced fluorescence scanner. The usefulness of this method was illustrated by analyzing severe acute respiratory syndrome (SARS) coronavirus RNA. The detection limit was 10(0) copies/microL. The results of the asymmetric multiplex nested reverse transcription-PCR were in agreement with the results of the microarray hybridization; no hybridization signal was lost as happened with applicons from symmetric amplifications. This reliable method can be used to the identification of other microorganisms, screening of genetic diseases, and other applications.
Zhang
Zhi-wei
ZW
Department of Biiological Sciences and Biotechnology, Tsinghua University, Beijing, China.
Zhou
Yi-ming
YM
Zhang
Yan
Y
Guo
Yong
Y
Tao
Sheng-ce
SC
Li
Ze
Z
Zhang
Qiong
Q
Cheng
Jing
J
D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D012680:Sensitivity and Specificity
D012367:RNA, Viral