線性探針技術(shù)原理

結(jié)核分枝桿菌

線性探針耐多藥檢測方法背景MDR/XDR疫情日趨嚴(yán)重現(xiàn)行的實驗室技術(shù)不能滿足臨床工作需要結(jié)核分枝桿菌耐藥機(jī)制研究清晰新診斷技術(shù)的快速發(fā)展2Rifampicin亞單位AABB抑制作用DNA依賴的

RNA聚合酶RNADNA模板突變rpoB利福平耐藥機(jī)制3rpoB基因突變MusserJ.M.(1995)Antimicrobialagentresistanceinmycobacteria:moleculargeneticinsights.ClinMicrobiolRev,8,496-514.4InactivationmutationmutationmutationOver-expressionOxidativestressAlkylhydrooxidasereductaseactivityOver-expressionDetoxificationINHresistanceINHPermiabilityalteration?oxyRahpCmabA(orf1)inhAInhANADH-dependentenoyl-ACPactivitykatGKatGAhpCCatarase-peroxydaseactivityINA/NADanalogueAccumulationofperoxideMycolicacidbiosynthesisINH異煙肼耐藥機(jī)制InactivationfurA5HazbónMH,etal..AntimicrobAgentsChemother.2006Aug;50(8):2640-9.katG

和

inhA突變6MTB基因突變與耐藥相關(guān)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耐藥檢測技術(shù)快速發(fā)展2000

INNO-LiPA(Belguim)2005

HainGenotypeLPA(Germany)2007

Genechip(CapitalBio)Verechip(Veredus)2010Genexpert(USA)20XX ?????LPAGenechipRT-PCR8HainLPAMTBC:分枝桿菌復(fù)合群內(nèi)部鑒定MTBCM:鑒定分枝桿菌復(fù)合群和非結(jié)核分枝桿菌MTBDRPlus：鑒定利福平和異煙肼耐藥性MTBDRsl：鑒定二線藥物耐藥性9MTBDRPlus原理基于多重PCR擴(kuò)增,擴(kuò)增產(chǎn)物通過反向雜交技術(shù)與預(yù)先固化在試紙條上的特異性探針雜交,通過檢測rpoB基因突變確定對利福平的耐藥性；通過檢測katG基因和inhA基因的啟動子區(qū)確定對異煙肼的耐藥性。標(biāo)本：痰標(biāo)本或菌株。檢測時間：1-2天報告結(jié)果。10PCR擴(kuò)增GeneSpecificsequenceAmplification11PathogenPCR擴(kuò)增21=222=423=824=1625=32:228=268,435,456229=536,870,912230=1,073,741,82412雜交MarkerControlMTBspecificprobeWildtypeprobesMutantprobesS1S2S3S4S5R2R4aR4bR5AmplifiedDNA3’5’AlkarynphosphataseStreptavidinBiotinSubstrateColour13

解鏈標(biāo)記的dsDNAPCR-產(chǎn)物化學(xué)變性標(biāo)記的ssDNA14擴(kuò)增產(chǎn)物-探針雜交探針DNA-帶45°C15嚴(yán)格漂洗不匹配！16顯色無色底物紫色底物DNA?STRIP?堿性磷酸酶鏈霉親和素1718結(jié)果判讀鑒定結(jié)核分枝桿菌復(fù)合群3個區(qū)域:rpoB,,katG

和inhA27條帶：6個對照條帶耐藥：WT缺失或MUT出現(xiàn)19rpoB區(qū)cagccagctgagccaattcatggaccagaacaacccgctgtcggggttgacccacaagcgccgactgtcggcgctggggcSerGlnLeuSerGlnPheMetAspGlnAsnAsnProLeuSerGlyLeuThrHisLysArgArgLeuSerAlaLeuG