Application of real-time PCR for analysis canine meat (Canis lupus familiaris) in goat’s satay for halal authentication study

Background: Canine meat (CM) is one of the non-halal meats prohibited for consumption by the Muslim community. Due to its low prices compared with beef, CM is typically used as meat adulterants in halal food-based products such as Satay and meatballs to get economic profits. Aim: The objective of this study was to design a novel species-specific primer in combination with real-time polymerase chain reaction for analysis of Canine’s DNA for halal authentication analysis. Methods: A Primer targeting the D-loop region of mitochondrial DNA was designed and subjected to a validation procedure by assessing some performance characteristics including specificity, amplification efficiency (E), sensitivity, repeatability, and linearity describing the correlation between the concentration of Canine’s DNA (x-axis) and quantification cycle (Cq) in y-axis. The designed primer was specific over other meat DNAs applying the annealing temperature (Tm) of 57.8°C. Results: The Real-Time Polymerase Chain Reaction (RT-PCR) method produced an acceptable amplification efficiency (E) of 109.7 with the coefficient of determination (R2) for the correlation between Cq and log DNA concentration of 0.999. The sensitivity of the developed method provides a limit of detection (LoD) value of 31.25 pg/µl. The precision of the analytical method is acceptable with a relative standard deviation value of 2. The method with the designed D-loop primer was successfully applied for the detection and quantification of Canine’s DNA in food products. There are no amplification profiles for Canine DNA in marketed goat’s satay products. Conclusion: RT-PCR combined with a novel primer targeting D-loop provides a specific and accurate analytical tool for the identification of CM for halal authentication studies. © 2025, Faculty of Veterinary Medicine, University of Tripoli. All rights reserved.