耐药结核分枝杆菌多重Taqman-MGB探针检测方法的建立

摘要

目的 建立耐药结核分枝杆菌的多重Taqman-MGB探针检测体系.方法 针对结核分枝杆菌抗结核一线药物突变位点[利福平: RpoB(526)/(531);乙胺丁醇: embB(306);异烟肼: KatG(315)、inhA(-15);链霉素: Rpsl(43)]设计并合成引物探针;构建耐药结核分枝杆菌野生型和突变型模板,对其进行梯度稀释;配制结核分枝杆菌耐药基因检测试剂,用不同类型模板进行检测;检测72例结核样本(耐利福平20株,耐异烟肼22株,耐链霉素1株,耐多药17株),并与罗氏药效试验结果比较.结果 该检测体系可同时检测6个结核突变位点,最低检测限为104拷贝/mL,对临床样本检测其总符合率为100％,一致性好(χ2=137.3,P＜0.001).结论 本研究建立了耐药结核分枝杆菌的快速检测方法,可较好应用于卫生检疫系统和临床实验室.%Objective To establish a multiple Taqman-MGB probe detection system for drug resistant Mycobacterium tuberculosis. Methods The primers and probes targeting mutation sites of Mycobacterium tuberculosis against tuberculosis first-line drug[rifampicin: RpoB ( 526) /( 531); ethambutol: embB ( 306); isoniazid: KatG ( 315) , inhA(-15); streptomycin: RpsL ( 43)]were designed and synthesized. Wild-type and mutant templates of drug resistant Mycobacterium tuberculosis were constructed and diluted. Drug resistance gene detection reagent of Mycobacterium tuberculosis was prepared, and tested by different templates. 72 tuberculosis samples ( rifampin resistance 20 strains; streptomycin resistance 22 strains; isoniazid resistance 1 strain; multiple drug resistance 17 strains) were analyzed by the reagent. The results were compared with Roche efficacy test. Results The detection system could simultaneously detect 6 tuberculosis mutation sites, and the minimum detection limit was 104 copies /mL. In clinical samples, the total compliance rate was 100％ with good consistency (χ2 =137. 3, P < 0. 001) . Conclusion This study establishes a rapid detection method of drug resistant Mycobacterium tuberculosis, which can be used for health quarantine system and clinical detection.