Abstract
Background:
Canine meat (CM) is one of the non-halal meats prohibited for consumption by the Muslim community. Due to low price compared with beef, CM is typically used as meat adulterants in halal-food based products such as Satay and meatballs to get the 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 validation procedure by assessing some performance characteristics including specificity, amplification efficiency (E), sensitivity, repeatability and linearity describing the correlation between 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:
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 developed method provides limit of detection (LOD) value of 31.25 pg/µL. The precision of analytical method is acceptable with relative standard deviation (RSD) value of 2%. The method with designed D-loop primer was successfully applied for 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 novel primer targeting D-loop provide a specific and accurate analytical tool for identification of CM for halal authentication studies.

Key words: D-loop Primer, Real Time-PCR, Canine meat, Goat’s Satay