Application of real-time fluorescent RT-PCR for quantitative detection of SARS-Coronavirus / 临床检验杂志

ABSTRACT

Objective To establish a real-time fluorescent quantitative RT-PCR for detecting SARS-Coronavirus (CoV) mRNA in gargling liquid and serum of SARS patients.Methods The assay is based on simplified nested fluorescent RT-PCR. Total RNA was extracted from gargling liquid and serum by using high performance system and reverse-transcription by antisense primer.The specific TaqMan probe was designed according to the published DNA sequence of SARS-CoV polymerase and labeled with FAM and TAMRA. Standard curves for SARS-CoV quantification were prepared with serial dilutions of the recombinant plasmid.Results The method is highly sensitive and specific. The sensitivity of assay was 1?105 copies/L. The positive rate of SARS-CoV mRNA in the gargling liquid of patients was 65.0% (26/40) and 11.5% (6/52) in suspected patients, respectively. SARS-CoV mRNA was not detectable in the gargling liquid from 40 healthy individuals. The positive rate of SARS-CoV mRNA in the serum of SARS patients was 25.6% (10/39). PCR amplification products of 6 suspected patients with SARS-CoV mRNA were confirmed by sequencing and the sequence data were consistent with that of BJ01 AY278488.Conclusions Real-time fluorescent quantitative RT-PCR should be a rapid, specific tool for early diagnosis of SARS-CoV infection.