摘要
目的评估ｒｐｏＢ基因突变检测在结核分支杆菌利福平耐药性测定中的应用价值。方法采用聚合酶链反应－冷单链构象多态性（ＰＣＲ－冷ＳＳＣＰ）方法对８７株结核分支杆菌临床分离株及有药敏结果的２２份相应肺结核患者痰标本进行分析。结果ＰＣＲ扩增ｒｐｏＢ基因的敏感性为１００ｐｇＤＮＡ及５０００个菌体，属于分支杆菌特异。所有分离菌的ｒｐｏＢ基因ＰＣＲ扩增均为阳性。采用套式ＰＣＲ可使扩增敏感性提高１００倍。与传统药敏试验方法相比，ＰＣＲ－冷ＳＳＣＰ对８７株结核分支杆菌临床分离菌利福平耐药性的检测敏感性和特异性分别为８９．６％及１００％。２２份涂阳培阳痰标本中套式ＰＣＲ扩增阳性的６份均为高度利福平耐药，其ＳＳＣＰ结果也与相应分离株的药物敏感性试验相符。结论ＰＣＲ－冷ＳＳＣＰ检测结核分支杆菌ｒｐｏＢ基因突变快速、简便、易行，适用于结核分支杆菌分离株利福平耐药性的快速测定。如进一步提高引物增特异性及敏感性。
ObjectiveToevaluatetheapplicabilityofdetectionofrpoBgenemutationinM.tuber-culosissusceptibilitytesting..Methods87M.tuberculosisisolatesand22sputumspecimensfrompa-tientswithactivepulmonarytuberculosisweredetectedbyPCR-SSCP.ResultshesensitivityofPCRforrpoBgeneamplificationwas100pgDNAand5000organisms.TherpoBgenecouldbedetectedinthealisolatestested.Incomparisonwithconventionalsusceptibilitytestingmethods,thesensitivityandspecificityofPCR-“cold”SSCPanalysisfordetectingrifampinresistancein87M.tuberculosisisolateswas89.6%and100%,respectively.Among22smear-andculture-positivesputumspecimens,only1(4.5%)waspositivebyPCR,however,6(27.3%)ofthemwerepositivebynested-PCR.The"cold"SSCPresultsofthese6specimenswerecorrespondingtothatofthesusceptibilitytesting..ConclusionsThePCR-"cold"SSCPdescribedherecaneasilyandrapidlydetectrifampinresistanceofM.tubercu-losis.Afterincreasingtheprimerspecificityandamplificationsensitivity,thetechniquemihgtbeusedforde-tectionofM.tuberculosisrifampinresistanceinclinicalspecimendirectly.
出处
《中华结核和呼吸杂志》
CSCD
北大核心
1996年第6期333-337,共5页
Chinese Journal of Tuberculosis and Respiratory Diseases
关键词
结核分支杆菌
RPOB基因
聚合酶链反应
多态性
MycobacteriumtuberculosisrpoBgenePolymerasechainreactionPoly-morphism
single-strandedconformationalDrugtolerance
