聚合酶链反应－单链构象多态性用于耐利福平结核分支杆菌rpoB基因突变的研究

ApplicationofPCR┐SSCPtechniqueindetectionofrpoBgenemutationinrifampin┐reslstantMy┐cobac┐teriumtuberculosis

目的研究结核分支杆菌ｒｐｏＢ基因突变及其与利福平（ＲＦＰ）耐药性的关系。方法以参考菌株结核分支杆菌Ｈ３７Ｒｖ为对照，用聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）方法分析了４０株结核分支杆菌临床分离株。ＰＣＲ－ＳＳＣＰ方法由ＰＣＲ扩增和扩增产物ＤＮＡ单链多态性分析组成。结果两对引物ＰＣＲ扩增产物分别为４１１和２５８ｂｐ，其敏感性分别为５ｐｇ／μｌ、５００个菌／ｍｌ和１ｐｇ／μｌ、５００个菌／ｍｌ；均为属特异性。４０株结核分支杆菌临床分离株２５８ｂｐ扩增片段ＳＳＣＰ图谱的特点：以参考菌株结核分支杆菌Ｈ３７Ｒｖ为对照，１０株敏感株均无区别；单耐ＲＦＰ或包括ＲＦＰ多种抗结核药３０株，除３株外，其余２７株ＳＳＣＰ图谱有明显的区别；检测阳性率为９０％，特异性为１００％。结论ＰＣＲ－ＳＳＣＰ方法可检测出耐ＲＦＰ结核分支杆菌ｒｐｏＢ基因突变；该基因是ＲＦＰ的药物靶编码基因，它的突变与ＲＦＰ耐药性有密切关系。

ObjectiveToevaluaterpoBgenemutationinrifampin-resistantMycobacteriumtuber-culosisanditsrelationshipwithrifampinresistance.MethodsFortyclinicalisolatesofMycobacteriumtuberculosiswereanalyzedbyPCR-SSCPtechnique,withH37Rvreferencestrainsascontrolgroup.Re-sultsThesensitivityofamplicationproductsof411bpand258bpwerefoundtobe5pg/μl,500organ-ismspermililiterand1pg/μl,500organismspermililiterrespectively.rpoBgenebelongstogenusspeci-ficity.CharacteristicsofSSCPgraphof258bpfragment:TensensitivestrainswerethesameasH37Rv.Thirtystrainsofrifampin-resistantormultidrugresistance,includingrifampin,werediferentfromH37Rvexceptforthreestrains.Positiveratewas90%,whilespecificity100%.ConclusionsResultsshowedthatPCR-SSCPtechniquecoulddetectrPOBgenemutation,whichmightassociatewithri-fampinresis-tanceandbehelpfultorapiddetectionandresearchofrifampin-resistantMycobacteriumtu-berculosis.