Novel duplex qPCR and LAMP assays Suitable for differentiating Mycobacterium bovis from other Mycobacterium tuberculosis complex species in MGIT sputum cultures

Multiple published qPCR and LAMP primer sets claiming specificity for M. bovis, M. tuberculosis or M. tuberculosis complex were selected, and several new primer sets were designed in-house. All primers were screened individually to assess detection sensitivity and specificity using DNA derived from 10-fold dilution series of M. bovis NCTC 1333 and M. tuberculosis H37Rv broth cultures (10⁶ to 10¹ CFU/ml). Then, the most sensitive and specific qPCR and LAMP primer sets for each target species were tested together, to check if they amplified successfully without detection sensitivity for either species being adversely impacted. Ultimately, both a duplex qPCR and a duplex LAMP assay were achieved. When a panel of gDNAs extracted from growth positive MGIT cultures was tested, both new tests correctly differentiated 17 M. bovis positive samples from M. tuberculosis (n=123), M. africanum (n=2) and M. abscessus (n=17) positive samples. The developed duplex assays take < 90 min after MGIT culture, so could expedite identification of the causative agent of TB infections.