结核分枝杆菌利福平和异烟肼耐药突变的快速分子诊断方法构建

Rapid molecular diagnostic method for rifampicin and isoniazid resistant mutations in Mycobacterium tuberculosis

目的 采用快速分子诊断方法中TaqMan荧光定量PCR技术,应用于利福平和异烟肼一线抗结核药物耐药基因的诊断中,评价其性能,建立高灵敏度、高特异度、高准确性并且快速的筛查方法,为结核分枝杆菌耐药性的诊断和治疗提供依据并探讨其应用价值,努力改善耐药结核病的检测和可行性。方法 本研究针对利福平ropB基因531位点突变和异烟肼katG基因315位点突变的特点,设计TaqMan-MGB引物和探针,通过反应条件优化,建立TaqMan荧光定量PCR方法;用克隆到pUC57载体上的阳性标准品及不同菌株评价该方法的特异性、灵敏度、重复性和精密度,采用甘油三酯、胆红素和血红素进行了干扰性实验。结果 TaqMan荧光定量PCR方法的检测限可达10copies/μl;在交叉干扰实验中,检测了5种常见呼吸道感染细菌国家标准菌株,均未检出,基因野生型和突变型探针特异性较好,不受其它基因干扰;批内、批间的CT值变异系数均小于5%,标准曲线R2=0.998,扩增效率均在90%以上;该反应迅速,检测时间为1h。结论 本研究建立的TaqMan荧光定量PCR方法有助于及时发现结核分枝杆菌耐药情况,对结核病的耐药检测与后续治疗具有重要意义。

Objective To use TaqMan fluorescence quantitative PCR technology in rapid molecular diagnosis method,applied in the diagnosis of drug resistance genes of first-line,evaluate its performance,establish a screening method with high sensitivity,high specificity,high accuracy and rapid screening method,provide basis for the diagnosis and treatment of mycobacterium tuberculosis and explore its application value,and strive to improve the detection and feasibility of drug-resistant tuberculosis.Methods In this study,Taq-Man-MGB primers and probes were designed to address the characteristics of the position 531mutation of ri fampin robB and the position 315mutation of isoniazid katG,quantitative PCR method for TaqMan fluorescence was optimized.The specificity,sensitivity,reproducibility and precision of the method were evaluated using positive standards and different strains cloned into the pUC57vector,and interference experiments were performed using triglycerides,bilirubin and heme.Results The detection limit of TaqMan fluorescence quantitative PCR method could reach 10copies/μl.In the cross-interference experiment,five national standard strains of common respiratory infection bacteria were detected,and the wild type and mutant probes were specific and not disturbed by other genes.The coefficient of variation between CT values was less than 5%,the standard curve R2=0.998,the amplification efficiency was above 90%.The reaction was rapid and the detection time was 1h.Conclusion The TaqMan fluorescence quantitative PCR method established in this study is helpful for the timely detection of drug resistance in Mycobacterium tuberculosis,which is important for drug resistance detection and subsequent treatment of tuberculosis.