中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2012年
7期
655-660
,共6页
褚美芬%严杰%钟雅文%吴亦斐%王欢%孙爱华
褚美芬%嚴傑%鐘雅文%吳亦斐%王歡%孫愛華
저미분%엄걸%종아문%오역비%왕환%손애화
结核分枝杆菌%异烟肼/利福平/耐药%基因突变%多聚合酶链反应-单链构象多态性/检测
結覈分枝桿菌%異煙肼/利福平/耐藥%基因突變%多聚閤酶鏈反應-單鏈構象多態性/檢測
결핵분지간균%이연정/리복평/내약%기인돌변%다취합매련반응-단련구상다태성/검측
Mycobacterium tuberculosis%Isoniazid/rifampin/resistance%Gene mutation%Multi-PCR-SSCP/detection
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.
目的 建立快速檢測結覈分枝桿菌異煙肼(INH)和利福平(RFP)耐藥相關基因katG、inhA和rpoB突變的多重聚閤酶鏈反應-單鏈構象多態性(multi PCR-single strand conformational polymorphism analysis,mPCR-SSCP)方法.方法 藥敏試驗檢測134株結覈分枝桿菌臨床菌株對INH和RFP的耐藥性.設計結覈分枝桿菌INH和RFP耐藥相關katG、inhA和rpoB基因PCR引物,建立mPCR-SSCP技術檢測上述菌株katG、inhA和rpoB基因的突變,同時採用PCR直接測序技術(PCR-DS)檢測上述基因片段突變情況,併對上述3種方法檢測結果進行分析和比較.結果 134株臨床菌株均含有katG、inhA和rpoB基因,其中42株(31.3％)對INH耐藥、45株(33.6％)對RFP耐藥.mPCR-SSCP和PCR-DS檢測結果顯示,92株INH敏感菌株katG和inhA基因均未髮生突變,檢測特異性均為100％；89株RFP敏感菌株中rpoB基因分彆有2株和1株檢測齣突變,檢測特異性分彆為97.8％和98.9％；42株INH耐藥菌株中分彆有33株和36株katG和/或inhA基因突變,檢測靈敏度分彆為78.6％和85.7％；45株RFP耐藥菌株中rpoB基因分彆有41株和43株髮生突變,檢測靈敏度分彆為91.1％和95.6％.結論 本研究建立的mPCR-SSCP能快速、簡便、特異,併有一定的敏感性檢測結覈分枝桿菌異煙肼和利福平耐藥相關基因katG、inhA和rpoB突變,具有臨床應用前景.
목적 건립쾌속검측결핵분지간균이연정(INH)화리복평(RFP)내약상관기인katG、inhA화rpoB돌변적다중취합매련반응-단련구상다태성(multi PCR-single strand conformational polymorphism analysis,mPCR-SSCP)방법.방법 약민시험검측134주결핵분지간균림상균주대INH화RFP적내약성.설계결핵분지간균INH화RFP내약상관katG、inhA화rpoB기인PCR인물,건립mPCR-SSCP기술검측상술균주katG、inhA화rpoB기인적돌변,동시채용PCR직접측서기술(PCR-DS)검측상술기인편단돌변정황,병대상술3충방법검측결과진행분석화비교.결과 134주림상균주균함유katG、inhA화rpoB기인,기중42주(31.3％)대INH내약、45주(33.6％)대RFP내약.mPCR-SSCP화PCR-DS검측결과현시,92주INH민감균주katG화inhA기인균미발생돌변,검측특이성균위100％；89주RFP민감균주중rpoB기인분별유2주화1주검측출돌변,검측특이성분별위97.8％화98.9％；42주INH내약균주중분별유33주화36주katG화/혹inhA기인돌변,검측령민도분별위78.6％화85.7％；45주RFP내약균주중rpoB기인분별유41주화43주발생돌변,검측령민도분별위91.1％화95.6％.결론 본연구건립적mPCR-SSCP능쾌속、간편、특이,병유일정적민감성검측결핵분지간균이연정화리복평내약상관기인katG、inhA화rpoB돌변,구유림상응용전경.
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,specificity and sensitivity,which shows a good prospect for application in clinic.
