Tracing the Pig and Cattle Origin in Processed Food and Feed Products Targeting Mitochondrial 12S rRNA Gene

Background: Species identification in commercially processed food and feed products is one of the important issues. This study was conducted to develop a genetic method for the detection of pig and cattle species in processed food and feed products using newly designed species-specific primers targeting mitochondrial 12S rRNA gene fragments. Methods: Two sets of specific primers were designed based on the 12S rRNA gene sequences of pig and cattle species from GenBank. The primers were validated by using the DNA extracted from nine different chordates, including pig, cattle, chicken, bata fish, bat, toad, African parrot, rat, and human origin. Annealing temperature ranging from 46-54°C for 30 seconds and template DNA 1:10 serial dilutions ranging from 10 to 0.00001 ng/µl were employed for primer annealing and sensitivity analysis. Samples were analyzed using optimized Polymerase Chain Reaction (PCR) conditions. Results: The most intense expected DNA bands of pig and cattle were produced at 50°C. Under that optimized annealing temperature pig and cattle-specific primers did not anneal with the DNA of other chordates. Total extracted DNA 0.001 ng and 0.01 ng of pig and cattle respectively containing the mitochondrial DNA (mtDNA) was successfully detected. Conclusion: These findings indicate that the newly designed primer pairs can be used to detect pig and cattle derivatives in various processed food and feed products.