The development of real-time polymerase chain reaction using species-specificprimer targeting cytochrome-b for identification of canine meat in mackerelmeatballs for halal authentication

The practice of mixing food using canine meat has been widely found. This practice is motivated by the low price of canine meat in the market. The purpose of this research was to design a species-specific primer (SSP) and develop a real-time PCR method for analyzing canine meat (Canis lupus familiaris) in mackerel meatballs intended for halal authentication. The design of SSP was carried out in silico using NCBI-Primer BLAST and IDT sites. The primer specificity analysis was performed on DNA canine and 8 animals namely mackerel, chicken, pig, goat, frog, cow, rat, and tree shrew using real-time PCR. Method validation is determined by sensitivity test, amplification efficiency, and repeatability test. The results showed that the Cytb-78 primer (forward: CAC TAA TCT TCT CTC TGC CAT CC, reverse: GAA TCG TGT TAG GGT TGC TTT G) which is specifically designed to amplify canine mitochondrial DNA with an optimum annealing temperature of 61.8°C. A sensitivity test was performed on 8 DNA dilution series with 10 dilutions resulting in the limit of detection (LoD) value of 1 pg/µL, an amplification efficiency (E) of 96.2%, and a coefficient of determination (R2 ) of 1. The repeatability test of 6 replicate DNA isolates of fresh meat resulted in a CV value of 0.72%. Analysis of the reference meatballs showed that Cytb-78 primer could amplify canine meat mixtures up to 10% concentration. The Cytb-78 primer that has been designed and the real-time PCR method that has been developed can be used for the analysis of canine meat (Canis lupus familiaris) in mackerel meatballs for halal authentication.