TaqMan-MGB荧光定量PCR快速检测结核分枝杆菌katG基因突变  

Detection on detecting katGmutation for rapidly diagnosis of Mycobacterium tuberculosiswith TaqMan-MGB real-time PCR

作　　者：潘爱珍[1,2] 赵秀芹[1] 张媛媛[1] 王晓萌[2] 柳正卫[2] 万康林[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所/传染病预防控制国家重点实验室,北京102206 [2]浙江省疾病预防控制中心,杭州310051

出　　处：《中国人兽共患病学报》2012年第1期14-18,23,共6页Chinese Journal of Zoonoses

基　　金：国家"十一五"重大传染病防治科技重大专项(2008ZX100/03-010-02)

摘　　要：目的建立一种特异、灵敏、快速检测结核分枝杆菌katG基因突变的TaqMan-MGB荧光定量PCR方法。方法根据结核分枝杆菌katG基因315位点突变的特点,设计一对特异性TaqMan-MGB探针和引物,通过反应条件优化,建立荧光定量PCR方法;用克隆到PMD18-T载体上katG基因阳性标准品及不同菌株来评价该方法的特异性、敏感性和重复性。结果灵敏度高,检测目的基因的最低检测下限10copies/μL,比常规PCR灵敏高100倍;特异性高,检测16株非结核分枝杆菌标准株和7种常见呼吸道感染细菌的标准株均为阴性;与测序法相比,野生型和突变型探针的敏感性和特异性均为100%。重复性好,批内批间CT值变异系数均小于1%。结论 TaqMan-MGB荧光定量PCR的方法能特异、灵敏、快速检测结核杆菌菌株katG315位点突变。The purpose of this study was to establish a TaqMan-MGB real-time PCR assay for rapidly,specificly,sensitively detecting the katG gene mutation of Mycobacterium tuberculosis（M.tuberculosis）.To conduct the design of TaqMan-MGB specific probes and primers,katG315 in M.tuberculosis was analyzed and compared.Through optimization of reaction conditions,the rapid assay to detection of M.tuberculosis resistant to isoniazid was founded;katG gene was cloned into PMD 18-T vector,and these positive standard strains and different strains was used to evaluated the specificity,sensitivity and repeatability;M.tuberculosis which were known sequencing results were detected the specificity and sensitivity of this assay.The minimum detection limit of the real-time PCR method in this study was 10 copies/μL,which was 100 times lower than that of conventional PCR.As the results of non-tuberculosis mycobacterium（NTM） and non-mycobacterium bacterium control strains tested with this method were negative,the specificity were 100%.The coefficients of variation for both intra-and inter-experimental were less than 1%,indicating remarkable repeatability.Compared with the results of sequencing,the sensitivity and specificity of wild-type and mutant-type probes were 100% respectively.The whole process which is rapidly detecting katG 315G→C in M.tuberculosis is sensitive,specific,easy,and short time.

关 键 词：结核分枝杆菌 KATG基因 TaqMan-MGB荧光定量PCR 

分 类 号：R378.91[医药卫生—病原生物学]