One-step multiplex PCR assay for identification of <i>Mycobacterium kansasii</i> complex species

Current methods used for the <i>Mycobacterium kansasii</i> complex (MKC) species identification lack the resolution to differentiate between individual MKC species and require time-demanding multiple steps and costly analyses. Herein, we present a novel one-step multiplex PCR assay that enables rapid and cost-effective species-level identification of MKC members, suitable for implementation in both clinical diagnostics and epidemiological investigations. The method was evaluated using 143 <i>Mycobacterium</i> strains, including 17 reference strains and 126 clinical or environmental isolates. It accurately identified the vast majority (98%) of strains, with only two atypical <i>M. kansasii</i> subtype IIB isolates misidentified as <i>M. persicum</i>. The calculated sensitivity and specificity of the assay were 98% and 100%, respectively.