Arefiev V. The system of indication and identification of Salmonella in feed based on multiplex-PCR.

Object of the study: The system of the screening and identification of Salmonella. Subject of the study: methods of the different Salmonella genetic material detection (Salmonella spp., S. Enteritidis, S. Typhimurium, S. Dublin, S. Gallinarum-Pullorum, S. Typhi), salmonella contamination monitoring system in objects of the veterinary-sanitary inspection. The thesis is devoted to the development of multiplex-PCR test-system for indication Salmonella genius and simultaneous identification of these major serological variations S. Enteritidis, S. Typhimurium, S. Dublin, S. Gallinarum-Pullorum, S. Typhi to detect microbial contamination of feed. For genius specific Salmonella detection and identification of S. Enteritidis, S. Typhimurium specific primers for genes invA, sefA and fliC were created, which flank region with length of 387, 299 and 433 bp in accordance. Optimization of the amplification protocol using constructed primers concerning temperature and timing parameters. This technique was used as the basis for the creation of test systems for the detection and typing of salmonella "Multi DNA test-Salm" (TR U 21.2-00497087-159), which according to commission testing is highly sensitive (98 %) and specific (100 %) for the detection of Salmonella DNA in feed. A screening feeds' study and their constituents regarding salmonella contamination was done. It was found that the diagnostic sensitivity of the test is 82.8 %, the diagnostic specificity - 99.1 % diagnostic efficiency - 97.0 %. Indices of values predictable positive results were estimated, which amounted to 92.8 %. The abovementioned demonstrates the importance of a variety of diagnostic PCR test systems. Feed contamination was detected in 10.4 % of all animal and vegetable origin samples, containing genetic material of S. Enteritidis (5.3 %), S. Typhimurium (0.8 %), S. Dublin (1.4 %), S. Gallinarum-Pullorum (1.6 %), and other Salmonella serogroups (1.4 %).