线性探针技术原理

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3rpoB基因突变MusserJ.M.(1995)Antimicrobialagentresistanceinmycobacteria:moleculargeneticinsights.ClinMicrobiolRev,8,496-514.4InactivationmutationmutationmutationOver-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

和

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-PCR8HainLPAMTBC:分枝杆菌复合群内部鉴定MTBCM:鉴定分枝杆菌复合群和非结核分枝杆菌MTBDRPlus：鉴定利福平和异烟肼耐药性MTBDRsl：鉴定二线药物耐药性9MTBDRPlus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GeneSpecificsequenceAmplification11PathogenPCR扩增21=222=423=824=1625=32:228=268,435,456229=536,870,912230=1,073,741,82412杂交MarkerControlMTBspecificprobeWildtypeprobesMutantprobesS1S2S3S4S5R2R4aR4bR5AmplifiedDNA3’5’AlkarynphosphataseStreptavidinBiotinSubstrateColour13

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1718结果判读鉴定结核分枝杆菌复合群3个区域:rpoB,,katG

和inhA27条带：6个对照条带耐药：WT缺失或MUT出现19rpoB区cagccagctgagccaattcatggaccagaacaacccgctgtcggggttgacccacaagcgccgactgtcggcgctggggcSerGlnLeuSerGlnPheMetAspGlnAsnAsnProLeuSerGlyLeuThrHisLysArgArgLeuSerAlaLeuG