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

M Thomas(1,2) B Gilbride(2) H Dane(2) D Fairley(3) Z Johnston(3) I R Grant(1,2)

1:School of Biological Sciences, Queen's University Belfast; 2:Rapid-Myco Technologies Limited, Belfast; 3:Mycobacteriology Laboratory, Royal Victoria Hospital, Belfast

Mycobacterium bovis causes zoonotic Tuberculosis (TB) in humans, mainly in developing countries. It differs from Mycobacterium tuberculosis, the predominant cause of human TB, by being pyrazinamide resistant, making it more difficult to treat. Otherwise, the species are similar phenotypically and genetically. Currently spoligotyping or whole genome sequencing are required to distinguish these two species after isolation from patient cultures. Our aim was to develop a duplex qPCR or LAMP assay that would permit rapid and easy differentiation of M. bovis from M. tuberculosis in positive MGIT cultures. We selected multiple published qPCR and LAMP primer sets claiming specificity for M. bovis, M. tuberculosis or M. tuberculosis complex, and designed several new primer sets in-house. All primers were screened 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 they could expedite identification of the causative agent of TB infections.

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