Development of a PCR test system for the indication of nontuberculosis mycobacteria in biological samples and the environment

Mycobacteria that are not part of the tuberculosis complex, non-tuberculosis mycobacteria (NTMB), are widespread in ecological reservoirs, in various species of domestic and wild animals, in soil and other environmental objects. A significant effect of NTMB has been established in the manifestation of nonspecific reactions to PPD-tuberculin in cattle which leads to unjustified slaughter of reacting animals, additional costs for differential diagnosis, loss of offspring and animal products, as well as huge costs for veterinary and sanitary measures. In this regard, the creation of a test system for screening environmental objects contaminated with NTMB and the identification of bacterial carrier animals is relevant. During the development of the test system, modern molecular biological methods were used, with the help of which primers and probes, species-specific for the most common NTMB, were identified, designed and synthesized. The temperature regime and the ratios of the components of the reaction mixture were optimized for the formulation of real time PCR. The developed test system was tested for specificity and sensitivity parameters using DNA of reference strains M. avium, M. paratuberculosis, M. fortuitum, M. intracellulare, M. smegmatis, M. scrofulaceum and M. kansasii. The results of research on the design of the test system formed the basis of the draft instructions for its use for the detection of NTMB DNA in biological materials and environmental objects by polymerase chain reaction (PCR) with hybridization-fluorescence detection in real time "NTMB PCR-RV".