基因芯片技术诊断耐多药结核病的临床应用研究

The application of gene chip in detecting the mutation of drug resistant gene in multi-drug resistant Mycobacterium tuberculosis strains

目的探讨基因芯片法检测耐多药结核分枝杆菌临床分离株耐药相关基因rpo B、kat G和inh A突变的特点,评估其临床应用价值。方法选取2013—2014年石家庄地区耐多药结核分枝杆菌临床分离株76株,采用基因芯片法检测rpo B、kat G及inh A基因耐药突变位点及频率,以比例法药敏结果为金标准,评价基因芯片法检测菌株耐药的准确度、灵敏度和特异度,Kappa检验评价基因芯片法和比例法药敏结果的一致性。结果在76株石家庄地区耐多药结核分枝杆菌临床分离株中,69株检测到kat G/inh A基因发生单一或联合突变,其中kat G基因优势突变位点为315,占89.9%(62/69),5.8%(4/69)存在inh A-15(C→T)突变,4.3%(3/69)发生kat G 315位点和inh A启动子区域的联合突变。73株检测到rpo B基因发生突变,优势突变位点为531,占64.4%(47/73);其次是526,突变率为15.1%(11/73);12.3%(9/73)发生516位点突变;1.4%(1/73)发生513位点突变;1.4%(1/73)发生533位点突变;另有5.5%(4/73)发生多位点的联合突变。以比例法药敏结果为金标准,基因芯片法检测利福平耐药的敏感度为96.1%(73/76),特异度为100.0%(50/50),总准确度为97.6%(123/126);检测异烟肼耐药的敏感度为90.8%(69/76),特异度为100.0%(50/50),总准确度为94.4%(119/126);基因芯片法检测耐多药结核的敏感度为86.8%(66/76),特异度为100.0%(50/50),总准确度为92.1%(116/126)。结论本地区耐多药结核分枝杆菌耐药相关基因优势突变类型为kat G基因315位点和rpo B基因531位点,基因芯片法可快速、有效诊断耐多药结核病。

Objective To understand the mutation characteristics of drug resistance-associated genes rpoB, katG and inhA in Mycobacterium tuberculosis （MTB） strains using gene chip method, and evaluate its clinical application value. Methods A total of 76 MTB strains were collected from Shijiazhuang area in 2013 to 2014. Gene chip was used to detect the mutations of rpoB, katG and inhA, and the L-J proportion drug susceptibility test was used as the gold standard to evaluate the overall concordance, sensitivity and specificity of gene chip. The consistency of microarray and phenotypic resistance was evaluated by Kappa test. Results Of all the 76 strains detected, 69 harbored mutations in katG/inhA. The predominant mutation site of katG was 315 codon with the mutation rate of 89.9%（62/69）, and 5.8%（4/69） carried mutations at inhA-15（C→T）, and 4.3%（3/69）carried combined mutations of katG 315 and inhA-15. The rpoB mutations were detected in 73 strains, of which 64.4%（47/73）carried mutations at codon 531, 15.1%（11/73）at codon 526, 12.3%（9/73）at 516 codon, 1.4%（1/73）at 513 codon, 1.4%（1/73）at 533 codon and 5.5%（4/73）had combined mutations. Compared with results from the L-J proportion method, the sensitivity, specificity and concordance rates of gene chip for RFP were 96.1%（73/76）, 100%（50/50）and 97.6%（123/126）. The sensitivity, specificity and concordance rates of gene chip for INH were 90.8%（69/76）, 100%（50/50）and 94.4%（119/126）. The sensitivity, specificity and concordance rates of gene chip for MDR-TB were 86.8%（66/76）, 100%（50/50） and 92.1%（116/126）. Conclusion The predominant mutation loci of MDR strains in Shijiazhuang area are katG315 and rpoB531. Gene chip is a fast and useful tool for clinical diagnosis of MDR strains.