A novel multiplex real - time PCR diagnostics assay for the identification and 1 differentiation of Mycobacterium tuberculosis , Mycobacterium canettii and 2 the Mycobacterium tuberculosis complex 3 4

26 Tuberculosis (TB) in humans is caused by members of the Mycobacterium 27 tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely 28 initiation of antibiotic treatment, while differentiation between members of the 29 complex may be important to guide the appropriate antibiotic treatment and provide 30 epidemiological information. 31 In this study, a multiplex real-time PCR diagnostics assay using novel 32 molecular targets was designed to identify the MTC while simultaneously 33 differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted 34 for the detection of members of the MTC, the wbbl1 gene was used for the 35 differentiation of M. tuberculosis and M. canettii from the remainder of the complex 36 and a unique region of the M. canettii genome, a possible novel “region of difference” 37 (RD), was targeted for the specific identification of M. canettii. 38 The multiplex real-time PCR assay was tested using 125 bacterial strains (64 39 MTC isolates, 44 non-tuberculosis mycobacteria (NTM) and 17 other bacteria). The 40 assay was determined to be 100% specific for the mycobacteria tested. Limits of 41 detection of 2.2, 2.17 and 0.73 cell equivalents were determined for M. 42 tuberculosis/M. canettii, the MTC and M. canettii, respectively, using Probit 43 regression analysis. 44 Further validation of this diagnostics assay, using clinical samples, should 45 demonstrate its potential for the rapid, accurate and sensitive diagnosis of TB caused 46 by M. tuberculosis, M. canettii and the other members of the MTC. 47