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目的 用基于多重PCR (multiplex PCR,mPCR)扩增子的二代测序(next-generation sequencing,NGS)技术(mPCR-NGS)检测MTB的耐药基因突变,并评估其检测石蜡包埋组织标本诊断耐药结核病的临床应用价值.方法 选取北京市昌平区结核病防治所2013年4月至2015年10月常规进行了利福平、异烟肼、乙胺丁醇、链霉素、卷曲霉素、卡那霉素、阿米卡星和氧氟沙星比例法检测且具有药敏试验结果的50株MTB临床分离株,包括42株耐药MTB和8株敏感MTB菌株.参考耐药基因数据库及相关文献,针对以上8种抗结核药物主要耐药基因及常见位点设计耐药基因组合,建立有效的检测方案.收集首都医科大学附属北京胸科医院病理科2017年11月至2018年9月耐药结核病患者55例的石蜡包埋组织标本,探针熔解曲线法检测结果为利福平、异烟肼、乙胺丁醇和氟喹诺酮类至少存在一种耐药相关基因突变.首先对已知表型药敏试验结果的MTB临床分离株进行mPCR-NGS检测,验证该方法检测纯菌样本的有效性,再进一步检测结核病患者石蜡包埋组织标本进行临床验证.主要评价指标为敏感度和特异度以及一致性.结果 以比例法为金标准,mPCR-NGS检测利福平、异烟肼、链霉素和乙胺丁醇的敏感度分别为95％ (38/40)、93％(27/29)、93％ (27/29)和72％(13/18),特异度分别为100％(10/10)、95％ (20/21)、100％(21/21)和94％(30/32);二线注射类药物卷曲霉素、卡那霉素和阿米卡星敏感度均为100％ (2/2,3/3,3/3),特异度分别为98％(47/48)、100％(33/33)和100％(47/47);氧氟沙星的敏感度为70％(7/10),特异度为100％(40/40).总符合率为94％,一致性检验Kappa值为0.87.纳入的55例石蜡包埋组织标本经探针熔解曲线法检测,28例利福平耐药,37例异烟肼耐药,13例乙胺丁醇耐药,17例氟喹诺酮类耐药.mPCR-NGS与探针熔解曲线法比较,利福平、异烟肼、乙胺丁醇和氟喹诺酮类的敏感度分别为100％(28/28)、95％(35/37)、100％(13/13)和100％(17/17);特异度均为100％(27/27,18/18,42/42,38/38).两种方法总符合率为99％,一致性检验Kappa值为0.98.结论 mPCR-NGS检测MTB临床分离株及结核病患者石蜡包埋组织标本耐药基因突变的敏感度、特异度和符合率较高,有望成为耐药结核病准确、快速的分子病理诊断新技术.“,”Objective To evaluate the use of multiplex PCR amplicon sequencing (mPCR-NGS) technology in detecting gene mutations related to drug resistance of Mycobacterium tuberculosis (MTB) in formalin-fixed paraffin-embedded tissue specimens,and to explore its clinical value in the diagnosis of drug-resistant tuberculosis.Methods Fifty clinical MTB strains isolated in the Changping District Tuberculosis Control Institute of Beijing from April 2013 to October 2015 with drug susceptibility test (DST) results of rifampicin,isoniazid,ethambutol,streptomycin,ofloxacin,capreomycin,kanamycin and amikacin available were recovered,including 42 drug-resistant strains and 8 drug-sensitive strains.The mPCR-NGS test was established to detect genes related to the 8 anti-tuberculosis drugs according to the previously published studies and databases.Fifty-five paraffin-embedded tissue specimens from drug-resistant tuberculosis patients were collected in the Department of Pathology,Beijing Chest Hospital,Capital Medical University during November 2017 to September 2018.All the specimens showed no less than one mutation in the gene regions related to drug resistance of any of the 4 drugs (rifampicin,isoniazid,ethambutol or fluoroquinolones) by probe melting curve assay.The effectiveness of mPCR-NGS test was evaluated on clinical MTB isolates using phenotypic DST as the reference.Clinical evaluation of mPCR-NGS test on formalin-fixed paraffin-embedded specimens from TB patients was performed using probe melting curve assay as the reference.The sensitivity,specificity and coincidence of mPCR-NGS were analyzed.Results Using phenotypic DST as the reference,the sensitivities of the mPCR-NGS for detecting drug-resistance of rifampicin,isoniazid,streptomycin,and ethambutol were 95％ (38/40),93％ (27/29),93％ (27/29),and 72％ (13/18),respectively;and the specificities were 100％ (10/10),95％ (20/21),100％ (21/21),and 94％ (30/ 32),respectively.The sensitivities for capreomycin,kanamycin and arnikacin were all 100％ (2/2,3/3,3/3),and the specificities were 98％ (47/48),100％ (33/33) and 100％ (47/47),respectively.The sensitivity and specificity of ofloxacin were 70％ (7/10) and 100％ (40/40),respectively.The total coincidence rate for the 8vdrugs was 94％,and the Kappa value was 0.87.The 55 paraffin-embedded tissue specimens included in this study were all tested by probe melting curve assays.Among them 28 were resistant to rifampicin,37 resistant to isoniazid,13 resistant to ethambutol,and 17 resistant to fluoroquinolones.Using the probe melting curve assay as the reference,the sensitivities of the mPCR-NGS for detecting resistant to rifampicin,isoniazid,ethambutol,and fluoroquinolones were 100％ (28/28),95％ (35/37),100％,and 100％,respectively;and the specificities were all 100％ (42/42,38/38).The total coincidence rate of the two methods was 99％,and the Kappa value was 0.98.Conclusions mPCR-NGS showed good sensitivities and specificities in detecting drug-resistant gene mutations both in clinical MTB isolates and paraffin-embedded tissue specimens,mPCR-NGS has the potential to be an accurate and rapid molecular pathological technology for diagnosis of drug-resistant tuberculosis.