One-step multiplex PCR assay for identification of Mycobacterium kansasii complex species

ABSTRACT Currently, the Mycobacterium kansasii complex (MKC) species identification involves PCR amplification, followed by sequencing or digestion of the amplicons. The purpose of this study was to develop a one-step multiplex PCR assay allowing for a fast and accurate identification of MKC species. A total of 158 Mycobacterium sp. genomes were searched for sites that would yield easily detectable amplicons of different sizes among the MKC species while producing no amplicons for other-than-MKC mycobacterial species. Three primer sets were designed and tested in vitro on seven reference strains, representing all MKC species. For the evaluation of the method, a total of 136 Mycobacterium sp. isolates were analyzed. The vast majority (96/98, 98%) of the tested MKC strains yielded a species-characteristic profile, consistent with in silico predictions. The only two isolates, which gave incongruent results, belong to atypical M. kansasii subtype IIB and were designated as M. persicum with a newly designed assay. No products were obtained for mycobacteria other than MKC. This study offers a new PCR-based method for identification of all MKC species. It involves a single-step protocol and yields reproducible and easily interpretable results. IMPORTANCE Current methods used for the Mycobacterium kansasii 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 Mycobacterium strains, including 17 reference strains and 126 clinical or environmental isolates. It accurately identified the vast majority (98%) of strains, with only two atypical M. kansasii subtype IIB isolates misidentified as M. persicum . The calculated sensitivity and specificity of the assay were 98% and 100%, respectively.