Molecular Biology and genetics Research Papers (original) (raw)

Background: Salmonellosis is one of the most dangerous diseases that is caused by Salmonella agents and has a wide spectrum of clinical manifestations - from asymptomatic to severe septic forms. In the majority of Salmonella infection cases, the enterica subspecies serovars are isolated from animals and humans. According to the FAO, 20% of poultry products in the world are contaminated with salmonella. Every year 21 million cases of typhoid fever are registered wherein 216 thousand are lethal. Traditional microbiological methods for Salmonella typing (cultivation) are usually time-consuming. This necessitates the development of modern methodology for food safety. Goal: Development of a multiplex PCR protocol enabling identification of Salmonella spp. and typing of Enterisa Salmonella Enteritidis, Salmonella Enterisa Typhimurium, Salmonella Typhi, Salmonella Dublin, and Salmonella Gallinarum. Methods & Materials: For amplification the following primers were used: Salmonella spp.: Salm3-Salm4 (Ferretti, 2001); Salmonella enteritidis: SentF-SentR (Agron, 2001); Salmonella typhimurium: StypF-StypR (O’Regan, 2008); Salmonella Typhi: StyphiF-StyphiR (Kumar, 2008); Salmonella Dublin: SdubF-SdubR; Salmonella Gallinarum: SgalF-SgalR (Akiba, 2011). Optimization of multiplex PCR protocol was performed according to Elnifro (Elnifro, 2000). Results: To determine the optimal PCR primer's annealing temperature the assays were performed at 58◦C, 60◦C, 63◦C, and 65◦C. The optimal amplification mode was determined to be as follows: Initial denaturation - 94◦C-2 min; Denaturation - 94◦C-45s; Annealing - 63◦C-45s; Extension - 72◦C-60s (40 cycles); Final extension - 72◦C-5 min. The optimal composition of the reaction mixture for multiplex PCR was: 10×DreamTaq Buffer-2,5ul, dNTP Mix, 2mM each-2,5ul, 25mM MgCl2-0,5ul, Primers 20pM, Template DNA-5,0ul, DremTaq DNA Polymerase-2,0ul Water, nuclease-free-3,5ul. The resulting protocol allowed for the detection of Salmonella spp. DNA in the samples, as well as the simultaneous typing of Salmonella Enterica Enteritidis, Salmonella Enterica Typhimurium, Salmonella Typhi, Salmonella Dublin, and Salmonella Gallinarum.The simultaneous amplification of all 6 expected fragments occurred in the multiplex PCR. Conclusion: The developed protocol is promising for the biological control of food safety, as well as in routine investigations.