To establish and validate a standardized protocol integrating traditional biochemical, immunological, and mass spectrometric techniques for the identification of Mycobacterium tuberculosis (M. tb) clinical isolates, and to screen candidate strains with typical characteristics.

Classical methods, including the heat-stable catalase test, PNB growth test, nitrate reduction test, Tween-80 hydrolysis test, urease test, MPT64 antigen detection, and MALDI-TOF MS, were used to identify clinical isolates obtained from sputum samples. Whole-genome sequencing was employed to analyze the genetic background and signature genes of the strains, and the broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of 16 anti-tuberculosis drugs.

Basic biochemical tests showed that the clinical isolates exhibited characteristics consistent with M. tb. MPT64 antigen colloidal gold detection was positive for all strains, and MALDI-TOF MS identification confirmed that all strains displayed M. tb features with a confidence score of 99.9%. Whole-genome sequencing analysis further revealed that the isolates were M. tb, belonging to Lineage 2 (Beijing family) and Lineage 4 (Euro-American lineage), with 100% homology in the 16S rRNA and hsp65 sequences compared with the reference genome H37Rv. MIC results showed that the isolates were susceptible to all tested first-line and second-line anti-tuberculosis drugs. Subculture stability observation showed that colony morphology, growth characteristics, and key phenotypic markers remained stable over successive passages.

Using a multidimensional verification strategy integrating traditional biochemical phenotyping, immunological markers, proteomics, and genomics, this study successfully isolated and characterized eight M. tb candidate strains with clear genetic backgrounds, well-defined phenotypic characteristics, and good in vitro subculture stability. These strains provide valuable resources for laboratory quality control and personnel training, and and serve as a reliable methodological foundation for future reference strain evaluation.