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

  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 high-throughput 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.