快速检测结核分枝杆菌异烟肼和利福平耐药相关基因mPCR—SSCP建立及应用

Establishment and application of multi-PCR-SSCP assay for rapid detection of isoniazid- and rifampin- resistance Mycobacterium tuberculosis isolates

目的建立快速检测结核分枝杆菌异烟肼（INH）和利福平（RFP）耐药相关基因katG、inhA和rpoB突变的多重聚合酶链反应-单链构象多态性（multi PCR-single strand conformational polymorphism analysis，mPCR-SSCP）方法。方法药敏试验检测134株结核分枝杆菌临床菌株对INH和RFP的耐药性。设计结核分枝杆菌INH和RFP耐药相关katG、inhA和rpoB基因PCR引物，建立mPCR-SSCP技术检测上述菌株katG、inhA和rpoB基因的突变，同时采用PCR直接测序技术（PCR—DS）检测上述基因片段突变情况，并对上述3种方法检测结果进行分析和比较。结果134株临床菌株均含有katG、inhA和rpoB基因，其中42株（31．3％）对INH耐药、45株（33．6％）对RFP耐药。mPCR-SSCP和PCR-DS检测结果显示，92株INH敏感菌株katG和inhA基因均未发生突变，检测特异性均为100％；89株RFP敏感菌株中rpoB基因分别有2株和1株检测出突变，检测特异性分别为97．8％和98．9％；42株INH耐药菌株中分别有33株和36株katG和／或inhA基因突变，检测灵敏度分别为78．6％和85．7％；45株RFP耐药菌株中rpoB基因分别有41株和43株发生突变，检测灵敏度分别为91．1％和95．6％。结论本研究建立的mPCR-SSCP能快速、简便、特异，并有一定的敏感性检测结核分枝杆菌异烟肼和利福平耐药相关基因katG、inhA和rpoB突变，具有临床应用前景。

Objective To establish multi-PCR-single strand conformational polymorphism analysis （mPCR-SSCP） for rapid detection of isoniazid （INH） and rifampin （RIF） resistance associated katG, inhA and rpoB genes of Mycobacterium tuberculosis isolates. Methods The INH- and RFP-resistance of 134 isolates was determined by using drug susceptibility test. The primers were designed for detecting INH and RFP resistance- associated katG, inhA and rpoB gene in the isolates by mPCR-SSCP. PCR-DS technique was applied to detect the mutations in katG, inhA and rpoB genes. All the results from different assays were subsequently analyzed as well as compared. Results All of the 134 tested isolates had katG, inhA and rpoB genes. Of the 134 isolates, 42 （31.3%） and 45 （33.6%） strains were INH- and RFP-resistant, respectively. The results of mPCR-SSCP and PCR-DS showed that all the 92 INH-susceptible isolates had no mutation in katG and inhA genes with 100% specificity. In the 89 RFP-susceptible isolates, 2 and 1 had mutation in rpoB genes confirmed by mPCR-SSCP and PCR-DS with 97.8% or 98.9% specificity, respectively. Among the 42 INH-resistance isolates, 33 and 36 strains had the mutations in katG and/or inhA genes due to the results of mPCR-SSCP and PCR-DS with 78.6% or 85.7% sensitivity, respectively. The results of mPCR-SSCP and PCR-DS also demonstrated that in the 45 RFP-resistance isolates, 41 and 43 strains had the mutations in rpoB gene with 91.1% or 95.6% sensitivity, respectively. Conclusion The mPCR-SSCP established in this study can be used to rapidly detect INH and RFP-resistance associated mutations in katG, inhA and rpoB genes of M. tuberculosis with convenience, specifici- ty and sensitivity, which shows a good prospect for application in clinic.