线性探针技术原理课件

结核分枝杆菌

线性探针耐多药检测方法背景MDR/XDR疫情日趋严重现行的实验室技术不能满足临床工作需要结核分枝杆菌耐药机制研究清晰新诊断技术的快速发展2Rifampicin亚单位AABB抑制作用DNA依赖的RNA聚合酶RNADNA模板突变rpoB利福平耐药机制3InactivationmutationmutationmutationOver-expressionOxidativestressAlkylhydrooxidasereductaseactivityOver-expressionDetoxificationINHresistanceINHPermiabilityalteration?oxyRahpCmabA(orf1)inhAInhANADH-dependentenoyl-ACPactivitykatGKatGAhpCCatarase-peroxydaseactivityINA/NADanalogueAccumulationofperoxideMycolicacidbiosynthesisINH异烟肼耐药机制InactivationfurA5HazbónMH,etal..AntimicrobAgentsChemother.2006Aug;50(8):2640-9.katG

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inhA突变6MTB基因突变与耐药相关性ZhangYetal.MechanismofdrugresistanceinMycobacteriumtuberculosis.ASMPress2005DrugActionmechanismGenesinvolvedRoleFrequencyofmutationinresistantMTBINHInhibitMAsynthesis;PotentialeffectsonDNA/lipids/CHO/NADmetabolismkatGProdrgconversion20-80inhADrugtarget15-43ndhINHactivitymodulator10ahpCMarkerofresistance10-15RIFInhibittranscriptionrpoBDrugtarget96PZACytoplasmacidification/de-energizedmembraneInhibitFAsynthesis?pncAProdrugconversion72-97fasIDrugtarget??EMBInhibitarabinogalactansynthesisembCABDrugtarget47-65SMInhibitproteinsynthesisrpsLDrugtarget52-59rrsDrugtarget8-21AK/KMInhibitproteinsynthesisrrsDrugtarget76FQNInhibitionofDNAgyrasegyrADrugtarget75-94gyrBParticipateindrugbinding?invitroIfrATransport?ETHInhibitMAsynthesisetaA/ethADrugtarget37inhADrugtarget56CSInhibitpeptidoglycansynthesisulrA/dadB?Drugtarget?7耐药检测技术快速发展2000 INNO-LiPA(Belguim)2005 HainGenotypeLPA(Germany)2007 Genechip(CapitalBio)Verechip(Veredus)2010Genexpert(USA)20XX ?????LPAGenechipRT-PCR8MTBDRPlus原理基于多重PCR扩增,扩增产物通过反向杂交技术与预先固化在试纸条上的特异性探针杂交,通过检测rpoB基因突变确定对利福平的耐药性；通过检测katG基因和inhA基因的启动子区确定对异烟肼的耐药性。标本：痰标本或菌株。检测时间：1-2天报告结果。10PCR扩增21=222=423=824=1625=32:228=268,435,456229=536,870,912230=1,073,741,82412

解链标记的dsDNAPCR-产物化学变性标记的ssDNA14扩增产物-探针杂交探针DNA-带45°C15严格漂洗不匹配！16显色无色底物紫色底物DNA•STRIP®碱性磷酸酶链霉亲和素1718rpoB区cagccagctgagccaattcatggaccagaacaacccgctgtcggggttgacccacaagcgccgactgtcggcgctggggcSerGlnLeuSerGlnPheMetAspGlnAsnAsnProLeuSerGlyLeuThrHisLysArgArg