Development of a one-step multiplex PCR assay for differential detection of major Mycobacterium species

Abstract

The prevalence of tuberculosis continues to be high, and nontuberculous mycobacteria (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, I 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 1) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750; 2) differentiate M. tuberculosis (MTB) from MTBC using RD9; 3) identify selectively the widespread MTB Beijing genotype by targeting mtbk_20680; and 4) detect simultaneously five clinically important NTM (M. avium, M. intracellulare, M. abscessus, M. massiliense, and M. kansasii) by targeting IS1311, DT1, mass_3210, and mkan_rs12360. The accuracy of the multiplex PCR assay was evaluated using 41 reference strains and 94 clinical isolates. Whole genome sequencing, spoligotyping and multi-locus sequence analysis were employed as the standard assays to evaluate the PCR assay in clinical MTB and NTM isolates, respectively. The initial PCR assay design demonstrated 100% sensitivity and 97.5% specificity for MTBC and 100% sensitivity and 100% specificity for the targeted NTM species. As the initial design misidentified two isolates of the MTB Central Asia Strain (CAS) lineage, I replaced rv0577 with RD750, a CAS-specific deletion, to discriminate CAS from MTBC. Reoptimization of the PCR assay resulted in 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTB, MTBC, MTB Beijing genotype, and major NTM species.