基于多重PCR-质谱微测序技术的结核分枝杆菌对一线治疗药物耐药性检测系统构建

Construction of Mycobacterium tuberculosis resistance detection system for first-line treatment drugs based on multiple PCR-mass spectrometry mini-sequence technology

目的构建一种基于多重聚合酶链式反应-质谱微测序技术的结核分枝杆菌耐药基因多位点检测方法和检测模块,实现结核分枝杆菌对一线抗结核药物的高通量快速检测,为结核病诊疗提供一种新型技术支撑。方法通过对5个单核苷酸多态性位点靶基因进行多重PCR扩增、单碱质量探针延伸及分子质量测定,同时完成16个突变位点、26种突变型的检测,实现结核分枝杆菌对利福平、异烟肼、吡嗪酰胺和乙胺丁醇等一线治疗药物抗性的通量检测。采用40例结核分枝杆菌样本、50例呼吸道感染患者咽拭子样本进行检测体系准确性及特异性验证。结果本研究构建了基于5重PCR-质谱联用的结核分枝杆菌对一线治疗药物的泛耐药检测方法,并开发了质谱配套检测系统。采用该系统,结核分枝杆菌对利福平、异烟肼、吡嗪酰胺、乙胺丁醇抗性检测的准确率和特异性均为100.00%,且具有7 h内完成96个样本的检测通量。结论本研究构建的方法有操作简便、低成本、高通量特点,耐药位点检测全面且准确,并具扩展性,可根据需要增添新突变位点,在结核分枝杆菌耐药性检测中具有良好应用前景。

Objective To establish a multi-locus detection method and detection module for Mycobacterium tuberculosis drug resistance gene based on multiplex PCR-mass spectrometry mini-sequencing technology and to realize the highthroughput rapid detection of first-line anti-tuberculosis drugs by Mycobacterium tuberculosis,which provide a new technical support for tuberculosis diagnosis and treatment.Methods Through multiplex polymerase chain reaction amplification,single base mass probe extension and molecular weight determination of 5 single nucleotide polymorphism locus target genes,and the detection of 16 mutation sites and 26 mutant types were completed at the same time,the flux detection of the resistance of Mycobacterium tuberculosis to first-line therapeutic drugs such as rifampicin,isoniazid,pyrazinamide and ethambutol was realized.The accuracy and specificity of the detection system were verified by using 40 samples of Mycobacterium tuberculosis and throat swab samples from 50 patients with respiratory infection.Results In this study,a pan-drug resistance detection method for first-line therapeutic drugs of Mycobacterium tuberculosis based on 5-plex PCR-mass spectrometry was constructed,and a mass spectrometry supporting detection system was developed.With this system,the accuracy and specificity of Mycobacterium tuberculosis resistance detection of rifampicin,isoniazid,pyrazinamide,and ethambutol were all 100.00%,and it had a detection throughput of 96 samples in 7 hours.Conclusion The method constructed has the characteristics of simple operation,low cost and high throughput,comprehensive and accurate detection of drug resistance sites,and scalability,and can add new mutation sites as needed,which has good application prospects in the detection and analysis of drug resistance of Mycobacterium tuberculosis.