Identification of species and optimization by using molecular methods in various treated meat

In this study, the detection of pork (Sus scrofa) meat mixed into beef (Bos taurus) in different proportions was made by SYBR Green-based real-time Polymerase Chain Reaction (SG-qPCR) method. Species-specific primers for porcine and bovine species were designed from the Adenosine triphosphate syntase subunit 8 and 6 gene regions on the mitochondrial DNA. It was observed that the porcine and bovine primers didn't cross-react with each other. Raw binary meat mixture samples were shaped as 6 cm diameter and 0.5 cm high meatballs with the help of a digital caliper. 10 pieces of meatballs were obtained from every 14 groups of meat samples. Autoclaving was applied to 5 meatball samples from each group at 121 oC at 15 psi for 30 minutes. DNA isolation from all raw and heat-treated meat samples was performed with a commercial kit. Primers designed specifically for porcine and beef were optimized for both raw and heat-treated meats. At the end of the optimization conditions for heat-treated meats; 15 μL reaction volume, 300 nmol each primer concentration, 50 ng /4 μL DNA concentration and 40 cycles, in raw meat samples; 10 μL reaction volume, 250 nmol each primer concentration, 50 ng/4 μL DNA concentration and 40 cycles were determined and analyzes were carried out in SG-qPCR. The study was conducted in 3 repetitions for each sample group and 3 techniques in each repeat. SG-qPCR products of samples belonging to all experimental groups were run on a 2% agarose gel to visualize target bands. In conclusion, the detection level and detection limit of pork in binary meat mixtures by using SG-qPCR were determined as 0.5% (w/w) and 0.25 ng/ μl in heat-treated meats, 0.1% (w/w) and 0.05 ng/μL in the raw meat mixture, respectively.