Multiplex Real-Time PCR Assay for Rapid Identification ofMycobacterium tuberculosisComplex Members to the Species Level

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Abstract
The species identification of members of theMycobacterium tuberculosiscomplex is critical to the timely initiation of both appropriate antibiotic therapy and proper public health control measures. However, the current commercially available molecular assays identify mycobacteria only to the complex level and are unable to differentiateM. tuberculosisfrom the closely relatedM. bovisandM. bovisBCG. We describe here a rapid and robust two-step, multiplex, real-time PCR assay based on genomic deletions to definitively identifyM. tuberculosis, M. bovis, M. bovisBCG, and other members of the complex. When tested against a panel of well-characterized mycobacterial reference strains, the assay was both sensitive and specific, correctly identifying all strains. We applied this assay to 60 clinical isolates previously identified asM. tuberculosiscomplex and found 57M. tuberculosisisolates and 3M. bovisBCG isolates from patients who had received intravesical BCG. Furthermore, analysis of 15 clinical specimens previously identified asM. bovisby spoligotyping revealed an isolate ofM. tuberculosisthat had been misidentified. We propose that this assay will allow the routine identification ofM. tuberculosiscomplex members in the clinical laboratory.

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