Establishment Of Self-quenched Fluorescent Quantitative PCR Method For Detection Of SARS Coronavirus

Objective: To establish a novel specific, sensitive and reliablequantitative RT-PCR method, based on the principle and technology oflight upon extension(LUX) fluorogenic primer , for rapid detection ofsevere acute respiratory syndrome (SARS) coronavirus. Methods: Choose the amplified target RNA sequence from the totalpublished severe acute respiratory syndrome(SARS) coronavirus completegenomes with the softwares of BLAST, Clustal x and RNAstructure. Thefluorogenic forward primer and the corresponding unlabeled reverseprimer were designed using LUX? Designer , and their specificity ,conservation and reliability were evaluated and optimized. cDNA for thetarget RNA sequence, which was transcribed in vitro with the template ofcloned plasmid from its SP6 promoter site, was cloned into the pGEM- Tvector with TA cloning protocol. We optimized the concentration of Mg2+ ,dNTP and primers in PCR reaction mixture by amplifying the RNAtranscribed in vitro above. Under optimal RT-PCR conditions, thesensitivity, quantitative linear range and variability of this method wereevaluated with the RNA transcribed in vitro as amplifying template. Results: The cloned fragment, which was sequenced, was identicalwith the corresponding sequence of the SARS coronavirus. OptimizedRT-PCR reaction system consisted of 3mmol/L Mg2+, 200μmol/L each ofdNTPs and 200nmol/L each of the primers. The optimal annealingtemperature was 55℃. The lowest detection limit in a PCR reaction tublewas 9.375 RNA molecules transcribed in vitro. A linear standard curvewith R2=0.993 was obtained, and the linear range was 1.5×102 copies/μlto 1.5×106 copies/μl. Intraassay coefficients of variation were 0.78% and1.1% for higher and lower concentration respectively; Interassaycoefficients of variation were 2.9% and 4.1% for higher and lowerconcentration respectively. Conclusion: A self-quenched fluorescent quantitative RT-PCRmethod was established, which was a rapid, specific and sensitive way toquantitatively detect severe acute respiratory syndrome (SARS)coronavirus RNA with a wide concentration range. This method is hopefulto be a novel cost-effective reliable tool to early quantitatively detectSARS coronavirus RNA.