Abstract: Objective To explore the matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS)platform for identifying Mycobacterium tuberculosis and detecting drug-resistant mutations and evaluate its application value. Methods MALDI-TOF-MS was used to detect three national reference strains, and the minimum detection limits for tuberculosis-specific identifying genes and drug-resistant mutations were determined. Respiratory tract clinical specimens of suspected tuberculosis patients were collected from January to September 2023 in the Tuberculosis Department of Kunming Third Peoples Hospital by non-random sampling method, including 120 sputum samples and 100 bronchoalveolar lavage fluid samples, with the content of 3-5 mL in each sample. Nucleic acid was extracted from all the samples, and the tuberculosis positive samples with Ct value≤32 were identified by qPCR kit for tuberculosis identification. The drug-resistant mutations of rifampicin(RIF), isoniazid(INH), ethambutol(EMB), moxifloxacin(MXF), streptomycin(SM)and pyrazinamide(PZA)were detected by MALDI-TOF-MS. The phenotypic drug susceptibility test(pDST)and third-generation sequencing(nanopore platfom)were used to detect the TB-positive samples, and the results were compared with those of MALDI-TOF-MS to verify its accuracy. Results The lowest detection limit of MALDI-TOF-MS was 60-100 CFU/mL for MTB identification genes IS6110 and ext_RD9, and 100-1 000 CFU/mL for drug-resistance mutations. A tolal of 132 tuberculosis positive samples with Ct value ≤ 32 were screened by qPCR. Compared with pDST results, the sensitivity, specificity and kappa value of MALDI-TOF-MS for detection of drug-resistant mutations were 93.75%, 100% and 0.96 for RIF, 98.33%, 90.28% and 0.88 for INH, 83.33%, 97.50% and 0.78 for EMB and 95.45%, 100% and 0.97 for MXF, respectively. Compared with nanopore sequencing, the sensitivity, specificity and kappa values of MALDI-TOF-MS for detection of drug-resistant mutations were 90.91%, 100% and 0.94 for RIF, 92.96%, 100% and 0.92 for INH, 92.86%, 100% and 0.96 for EMB, 95.45%, 100% and 0.97 for MXF, 95.74%, 100% and 0.97 for SM, 66.67%, 100% and 0.80 for PZA, respectively. Conclusion MALDI-TOF-MS can be used as an effective method for rapid detection of Mycobacterium tuberculosis drug-resistant mutations because its results were highly consistent with those of pDST and nanopore sequencing.

Key words: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Mycobacterium tuberculosis, Drug resistance, Phentypic drug susceptibility test, Nanopore sequencing