Development and evaluation of multiplex real-time PCR for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern China

Background: (MTB) and non-tuberculous mycobacteria (NTM) have similar clinical symptoms but require different treatments. Rapid and accurate identification of MTB and NTM is essential for proper patient management and treatment. Methods: in clinical samples. The specificity and limit of detection (LOD) were tested using standard strains and clinical isolates. The accuracy of the Multiplex PCR was validated with DNA from 228 known clinical samples confirmed by Targeted next-generation sequencing (tNGS). Additionally, 901 consecutive clinical samples were assessed to evaluate the Multiplex PCR's diagnostic performance in detecting mycobacteria in pulmonary and extrapulmonary samples. Results: < 0.01), with a specificity of 96 %. Conclusions: The Multiplex PCR demonstrated excellent diagnostic performance in both pulmonary and extrapulmonary samples, offering a low-cost, rapid identifying tool for major pathogenic mycobacteria in eastern China.