Sensitive Detection of SARS Coronavirus RNA by a Novel Asymmetric Multiplex Nested RT-PCR Amplification Coupled With Oligonucleotide Microarray Hybridization

Abstract

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.