DNA微阵列法检测结核分枝杆菌对利福平和异烟肼的耐药性

Detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis by DNA micro-array method

目的探讨DNA微阵列法在结核菌耐药基因快速检出中的应用价值。方法应用DNA微阵列法定性检测来源于临床疑似结核病患者痰液样本,检测利福平(RFP)和异烟肼(INH)的3个耐药相关基因rpo B、kat G及inh A基因启动子的野生型及不同突变型;采用BACTECTMMGIT 960培养仪进行结核分枝杆菌(M.tuberculosis)培养,阳性液体培养物用改良罗氏比例法进行药物敏感试验。结果 174份M.tuberculosis核酸阳性痰液标本中15份rpo B突变型,突变频率最高的位点是531,突变率66.7%;17份kat G/inh A突变型,其中12份是kat G位点的单一突变,突变率为70.6%。对照68例改良罗氏比例法药敏结果,DNA微阵列法检测RFP、INH耐药的符合率均达90%以上。结论 DNA微阵列法可快速准确地检测大部分疑似结核病临床样本的rpo B、kat G和inh A基因突变,可用于临床耐药性的检测,指导临床用药,并在临床诊断中推广应用。

Objective To explore the application value of DNA micro-array method for quick detection of drug resistant genes of Mycobacterium tuberculosis（M.tuberculosis）.Methods DNA micro-array method was adopted to detect the M.tuberculosis in sputum samples of clinically suspicious TB patients.The wild genotypes and different mutant genotypes of promoters of three genes relevant to resistance of rifampicin（RFP） and isoniazid（INH）,i.e.rpoB,katG,and inhA,were detected.BACTECTM MGIT 960 system was used to cultivate M.tuberculosis and the modified Roche proportion method was adopted to perform drug susceptibility for positive liquid cultures.Results Among 174 positive liquid cultures,15 of them were rpoB mutant genotype.The mutation frequency of locus 531 was the highest and mutation rate was 66.7%.Seventeen cultures were katG/inhA mutant genotype and 12 of them were single mutation of katG with the mutation rate of 70.6%.Compared with the results of 68 cultures detected by the modified Roche proportion method,the detection rate of DNA micro-array method for detecting RFP and INH resistance was more than 90%.Conclusion DNA micro-array method can quickly and reliably detect the gene mutation of rpoB,katG,and inhA in most clinical samples of suspicious TB patients.This method can be used to detect clinical drug resistance and guide clinical medication and diagnosis.