Simultaneous Detection of Bovine and Porcine DNA in Meat Products using Duplex Real-Time PCR Method for Halal Authentication

Assuring the halal status of meat based food products requires analytical methods that are sensitive and specific and can detect cross contamination. This study aims to develop and validate a SYBR Green based duplex real-time PCR method for the simultaneous detection of bovine (Bos taurus) and porcine (Sus scrofa domesticus) DNA in meatball products. DNA was isolated using the Geneaid DNA Isolation Kit, analyzed qualitatively by electrophoresis, and quantitatively by Nanodrop spectrophotometer. The annealing temperature was optimized at 50–60°C. The specificity test showed a Tm of 75–76°C for the Beef primer and 81.5°C for the porcine primer, with no cross amplification or dimers in the NTC. The duplex method detected both targets simultaneously with an LOD of 0.0005 ng/ µL. The standard curve had a slope of 2.79, efficiency of 128%, and R² of 0.932. Repeatability testing showed an average RSD of 2.829% (meeting the RSD ≤ 25% requirement). Analysis of meatball samples from the market showed bovine DNA without porcine DNA contamination. These results prove that the duplex real-time PCR method has high sensitivity and specificity for halal product verification.