反向线性点杂交方法检测embB基因突变在筛查结核分枝杆菌耐多药株的研究  

Research of detecting embB mutation to screen MDR-TB by reverse line blot assay

作　　者：张楠[1] 胡继红[1] 高振祥[1] 张然[1] 

机构地区：[1]卫生部北京医院卫生部临床检验中心,北京100730

出　　处：《中国卫生检验杂志》2011年第3期537-539,542,共4页Chinese Journal of Health Laboratory Technology

基　　金：国家自然科学基金面上项目(30771853)

摘　　要：目的:利用反向线性点杂交(Reverse Line Blot assay,RLB)技术建立检测embB基因突变筛查结核分枝杆菌耐多药株(指至少对异烟肼和利福平两种以上药物产生耐药的结核分枝杆菌,multi-drug resistant tuberculosis,MDR-TB)的方法。方法:采用比例法药敏试验检测301株结核分枝杆菌临床分离株的耐药表型,分别用测序及反向线性点杂交方法检测embB基因突变位点,并用比例法药敏试验作为表型检测对照方法,测序作为基因型检测对照方法,对RLB方法筛查耐多药株进行评价。结果:经表型药敏检测耐多药株占57.9%,非耐多药株占25.2%,全敏感株占16.9%。经测序检测94株发生embB Met306突变,其中89.4%(84/94)的突变发生在耐多药株1,0.6%为非耐多药株。Met306突变率在乙胺丁醇(Ethambutol,EMB)耐药组46.9%与乙胺丁醇敏感组47.8%间差异无统计学意义(χ2=0.01,P>0.05),而突变率在耐多药组为48.3%与非耐多药组13.2%间差异有统计学意义(χ2=48.53,P<0.01)。用反向线性点杂交检测embB基因突变位点与测序法相比敏感度为96.8%,特异度为99.0%,二者一致率达98.3%。用反向线性点杂交检测耐多药株与表型检测方法相比敏感度为48.3%,特异度为88.2%,二者一致率为60.4%。结论:反向线性点杂交方法检测em-bB基因突变可在常规检测中替代测序法快速筛查结核分枝杆菌耐多药株。Objective：To establish a new rapid method to screen the MDR-TB by detecting the embB mutation based on RLB.Methods：Totally of 301 isolates collected in China were tested by drug sensitive test（DST） to detect the phenotype resistance.Sequencing and RLB technique were applied to detect embB mutation genotype resistance.Meanwhile,we evaluated the results of detecting embB mutation to screen MDR-TB based on RLB by phenotype method and genotype method.Results：By genotype detecting 57.9% was MDR-TB,25.2% was nonMDR-TB,16.9% was whole-susceptibility.By sequencing detecting,embB mutation were detected in 94 isolates,in which 89.4%（84/94） were MDR-TB,10.6% were non MDR-TB.The embB mutation rate between the EMB-resistance（46.9%） and EMB-susceptibility（47.8%） was no significance（χ2=0.01,P0.05）.But between the MDR-TB（48.3%） and nonMDR-TB（13.2%） statistic discrepancy was tested（χ2=48.53,P0.01）.Comparing with sequencing method to detect embB mutation,the sensitivity of RLB is 96.8%,the specificity is 99.0%,and the coincidence rate is 98.3%.Comparing with phenotype detecting to screen MDR-TB,the sensitivity of RLB is 48.3%,the specificity is 88.2%,and the coincidence rate is 60.4%.Conclusion：In routine method,detecting the embB mutation by RLB can replace the sequencing method to screen the MDR-TB.

关 键 词：结核分枝杆菌 EMBB基因 耐多药 反向线性点杂交 

分 类 号：R378.911[医药卫生—病原生物学]