不明原因肺炎监测中SARS-CoV实时荧光RT-PCR检测方法的建立

目的 建立SARS-CoV的TaqMan探针实时荧光RT-PCR检测方法并进行评估,为不明原因肺炎监测中SARS-CoV感染的排查提供实验室检测依据.方法利用SARS-CoV的1b基因和NP基因的特异性序列设计引物和探针,两条探针5'端标记FAM,3'端标记TAMRA.与国家流感中心发布的高致病性人禽流感H5N1的实时...

Establishment of the Real-time RT-PCR for Detection of SARS-CoV during the Surveillance of Unexplained Pneumonia

Objective To develop and evaluate a real-time RT-PCR method for detecting SARS-CoV, based on TaqMan hybridization probe technology in order to provide a laboratory diagnosis for the examination of SARS-CoV infection during the surveillance of unexplained pneumonia. Methods Two pairs of primers and probes were designed to identify 1b and NP gene of SARS-CoV respectively. The two probes were 5'end labeled with FAM and 3'end labeled with TAMRA. The PCR reaction conditions were optimized according to the reaction conditions for detecting H5N1 which was set up by the National Influenza Center. Different concentration of plasmid DNA containing the target gene, 12 kinds of other respiratory viruses, mycoplasma pneumoniae and legionella pneumophilia were tested using this method to evaluate the specificity, sensitivity and reproducibility of the assay. Results All kinds of the viruses, mycoplasma pneumoniae and legionella pneumophilia tested were negative. The sensitivity of this assay for 1b gene was 10-9μg/ml DNA/reaction and for NP gene was 10 -7μg/ml DNA/reaction. The coefficients of variation (CV) value were 0.2% -0.9% during the reproducibility test. The whole process takes 2.5h including the extraction of RNA from the sample, and could be completed in the same machine, under the same condition with the detection of H5N1.Conclusion This real-time RT-PCR setting up based on TaqMan probe is a specific, rapid and sensitive method for detecting SARS-CoV. The establishment of this method will provide a strong support for quick examination of SARS-CoV infection during the unexplained pneumonia surveillance.