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.