Development of a multiplex PCR for detection of pathogenic Mycobacterium orygis in cattle tissues harboring tuberculous-like lesions: a pilot study.

Mycobacterium orygis, a member species of Mycobacterium tuberculosis complex (MTBC), is emerging as a major threat to the control of zoonotic tuberculosis. The lack of low-cost diagnostic assays to differentiate M. orygis from other MTBC members has created a critical data gap in estimating its true burden in human and animal populations, especially in the Asian subcontinent. In this study, a multiplex PCR assay was developed to distinguish M. orygis from other MTBCs, based on two M. orygis-specific non-synonymous point mutations in the mbtG and fadD23 genes. The assay exhibited a limit of detection (LOD) of 32 pg for M. orygis DNA and demonstrated high specificity by amplifying a 434 bp IS1081 gene fragment from common members of the MTBC, including M. orygis. Also, 240 bp and 181 bp fragments of the mbtG and fadD23 gene fragments were amplified exclusively from M. orygis. Conversely, the assay showed no amplification with non-tuberculous Mycobacterium or other non-mycobacterial pathogens. Furthermore, tuberculous-like lesions in the different tissues (n = 135) of slaughtered cattle were tested for the identification of M. orygis. An established TaqMan real-time PCR assay carried out to validate the result by targeting regions of difference in the M. orygis genome of the assay showed high accuracy of the in-house developed multiplex PCR.