不明原因肺炎监测中SARS—CoV实时荧光RT-PCR检测方法的建立  

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

作　　者：吕燕宁[1] 彭晓旻[1] 王小梅[1] 张代涛[1] 崔淑娟[1] 郭婧[1] 杨鹏[1] 黄芳[1] 王全意[1] 

机构地区：[1]北京市疾病预防控制中心,100013

出　　处：《国际病毒学杂志》2011年第1期15-22,共8页International Journal of Virology

摘　　要：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Ib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的实时荧光RT—PCR检测方法进行整合，优化了反应体系及反应条件，并对不同浓度的含有目的基因的质粒以及12种其他呼吸道病毒、肺炎支原体和嗜肺军团杆菌核酸进行检测，验证该方法的特异性、敏感性和重复性。结果该方法所检测的12种呼吸道病毒及肺炎支原体和嗜肺军团杆菌均为阴性；对目的基因的检测灵敏度1b基因达10^-7μg/mlDNA／反应，NP基因达10^-9μg／mlDNA／反应；重复性实验中，变异系数为0．2％-0．9％；从样本的核酸提取至检测完成仅需2．5h左右，并可与高致病性人禽流感H5N1的检测同台仪器同条件下完成。结论本文建立的TaqMan探针实时荧光RT-PCR是一种检测SARS-CoV的特异、快速、敏感的方法。该方法的建立将有益于今后开展不明原因肺炎监测以及应急临床标本中SARS—CoV感染的排查和快速检测。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 1 b 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, mycoplas- ma pneumoniae and legionella pneumophilia were tested using this method to evaluate the specificity, sensi- tivity 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 1 b gene was 10^-9μgg/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 HSNI. 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.

关 键 词：SARS—CoV 不明原因肺炎 实时荧光RT—PCR 

分 类 号：R392.11[医药卫生—免疫学]