Objective To evaluate the sensitivity and specificity of Tag Array in detecting the clinical isolates of bone and joint tuberculosis.Methods Twenty-four strains of Mycobacterium tuberculosis having sequence differences in extracted plasmids of mutant strains.The plasmid was diluted into different concentrations,and then multiplex PCR amplification was performed to analyze the sensitivity and specificity of the chip system.A total of 187 clinical isolates were collected from patients with bone and joint tuberculosis.Among them,there were 50 strains of sensitive bacteria,and 137 strains of drug-resistant.Designed,optimized and prepared chip inspection system,sequencing and phenotypic drug susceptibility results were analyzed to evaluate the sensitivity and specificity of the gene chip.Results The mutants in accordance with sequencing result in the sets with 1 × 103copy/μl of template concentration and above;while 29 sets showed false negative result in the sets with 1 × 102 copy/μl.Among the 126 rifampicin phenotype drug resistance strains,119 strains appeared mutation in rpoB gene.In the rifampicin phenotype sensitivity strains,9 strains of rpoB gene showed mutation,the sensitivity was 94.40％,and specificity was 86.76％.Among the 118 isoniazid phenotype drug resistance strains,109 strains appeared mutation in relevant drug resistance gene locus,the sensitivity was 92.37％,and specificity was 81.16％.Among the 44 ethambutol phenotype drug resistance strains,27 strains appeared mutation in locus embB306,in the ethambutol phenotype sensitivity strains,the mutation in locus embB306 was detected in 6 strains,the sensitivity was 61.36％,and specificity was 95.80％.Among the 102 levofloxacin phenotype drug resistance strains,81 strains appeared mutation in gyrA gene,4 of the sensitivity strains were detected mutation in gyrA gene,the sensitivity was 79.41％,and specificity was 95.29％.Among the 112 streptomycin phenotype drug resistance strains,the chip detected 101 strains appeared mutation,the sensitivity of chip detection was 90.17％,and specificity was 84.00％.Since amikacin,capreomycin and kanamycin are in cross drug resistance,among the 67 phenotype drug resistance strains,52 strains appeared mutation in relevant drug resistance gene locus,its sensitivity was 79.10％ and specificity was 90.83％.Conclusion Tag Array chip can achieve rapid,accurate and high-throughput detection of tuberculosis resistance,which has important clinical significance and feasibility.%目的 评价Tag Array结核耐药基因检测芯片检测骨关节结核临床分离株的敏感度和特异性.方法 在北京疾控中心获取已进行测序的24株不同突变型的结核杆菌菌株并进行质粒提取.进行浓度检测,并将不同质粒分别稀释成1×102拷贝/μ1、1×103拷贝/μ1、1×104拷贝/μl、1×105拷贝/μl、5×105拷贝/μl、1×106拷贝/μl后进行多重PCR扩增及芯片杂交,分析该芯片检测样本的敏感度和特异性,评估其在临床实际检测中的可行性.从重庆市公共医疗卫生救治中心获取187株骨结核临床分离株样本,其中敏感菌50株,耐药菌137株.设计、优化和制备芯片检测体系,对187株结核分枝杆菌临床分离株进行药敏检测,对照测序结果和表型药敏结果分析芯片的灵敏度、特异性.结果 在1×103拷贝/μl及以上组均显示出与测序结果一致的突变型,在1×102拷贝/μl组有29株出现了假阴性结果.126株利福平表型耐药株中,芯片检测出119株rpoB位点突变;表型敏感株中,检测出9株rpoB位点突变,与表型药敏株结果比较,敏感度为94.40％;特异性为86.76％.118株异烟肼表型耐药株中,芯片检测共109株出现耐药基因位点突变,其敏感度为92.37％;特异性为81.16％.44株乙胺丁醇表型耐药株中,27株在embB306位点上突变;表型敏感株中,有6株检测到embB306位点突变,其敏感度为61.36％;特异性为95.80％.102株喹诺酮类药物表型耐药株中,81株gyrA基因突变;敏感株中有4株检测到gyrA突变,敏感度为79.41％;特异性为95.29％.112株链霉素表型耐药株中,芯片检测出101株突变,其敏感度为90.17％;特异性为84.00％.二线抗结核药物(阿米卡星、卷曲霉素、卡那霉素)具有交叉耐药性,在67株表型耐药株中,52株存在突变;在表型敏感株中,有11株rrs1401突变,敏感度为77.61％,特异性为87.50％.结论 通过Tag Array结核耐药基因检测芯片可实现结核耐药快速、准确、高通量检测,具有重要的临床意义及可行性.
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