TDI-FP技术检测耐乙胺丁醇结核分支杆菌embB306点突变

Detect embB306 gene mutation associated with EMB resistance of M. tuberculosis by TDI-FP technology

目的 应用TDI FP技术分析结核分支杆菌embB 30 6点突变与乙胺丁醇耐药性的关系 ,并建立一种准确、快速的诊断结核分支杆菌乙胺丁醇耐药的新方法。方法 对临床 82例耐多药结核病患者所感染的结核分支杆菌菌株进行常规培养和药敏实验 ;提取结核分支杆菌DNA ,并PCR扩增 2 0 5bp的embB基因片段 ,消化降解掉PCR产物中残余的dNTPs和引物 ,进行模板指导的荧光标记终止碱基掺入反应 ;应用Victor2测定反应产物的荧光偏振值 ,分析所有样品的embB基因 30 6位点的基因型 ;对分析结果进行测序验证。结果 5例乙胺丁醇高度耐药患者中有 3例所感染的结核分支杆菌发生embB 30 6的ATG→GTG突变 ;4 7例乙胺丁醇低度耐药患者中有 15例为embB 30 6突变和未突变的混合感染 ;30例乙胺丁醇敏感患者的结核分支杆菌未发现embB 30 6突变。测序结果与实验相符合。结论 embB 30 6突变与结核分支杆菌对乙胺丁醇产生耐药性密切有关 ;应用TDI FP技术可以准确、快速简便检测embB 30 6突变 ,在结核分支杆菌耐乙胺丁醇的临床诊断中具有潜在的应用前景。

Objective To study the relationship between the mutation of emb B 306 and ethambutol(EMB) resistance of M. tuberculosis by TDI-FP, and develop a new method that can detect EMB-resistant M.tuberculosis precisionly and fastly. Methods The M. tuberculosis resisted to EMB from 82 tuberculosis patients were cultured on culture medium with different EMB concentration; Extract DNA of all M. tuberculosis samples respectively, and amply 205 bp fragment of emb B gene by polymerase chain reaction(PCR). After removing the excess primers and dNTPs in PCR products, template-directed dye-terminator incorporation reaction was performed; Measure Fluorescence polarization value using Victor2 multilabel counter, and then investigate the genotype of the mutation site in 306 condon of emb B gene of all samples; Verify the genotype of part of samples by sequencing. Results There were 3 out of 5 isolates of M. tuberculosis with high EMB-resistant showed mutation of ATG→GTG in 306 condon of emb B gene, in 47 isolates of M. tuberculosis with low EMB-resistant, 15 were mixing infection, none of 30 susceptible isolates of M. tuberculosis was found mutation in 306 condon of emb B gene. Sequence analysis shows the same results. Conclusion The ATG→GTG mutation in 306 condon of emb B gene showed high relativity with EMB-resistance of M. tuberculosis. TDI-FP technology is a reliable, easy and highthrought method to detect mutation of emb B gene, and it will be a useful technique for EMB-resistance diagnosis in the future.