Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species

The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the complex (MTBC) and NTM using or RD750, (ii) differentiate () from MTBC using RD9, (iii) selectively identify the widespread Beijing genotype by targeting , and (iv) simultaneously detect five clinically important NTM (, , , , and ) by targeting IS, DT1, , and An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 and 10 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, , Beijing genotype, and major NTM species.