Qualitative and Quantitative Analysis of Canine (Canis lupus familiaris) Meat in Meatballs for Halal Authentication Study using Real-time Polymerase Chain Reaction

摘要

Canine meat (CM), one of non-halal meats having low price value, may be used as substitute or meat adulterant in beef meatballs by unethical producers to get economic benefits. This study was intended to design and to validate specific primer targeting on cytochrome-b gene for detection of CM in meatballs using real-time polymerase chain reaction (real-time PCR). The primer was in silico designed and was subjected to specificity test against DNAs extracted from several meats. The evaluation of other validation parameters for quantitative analysis was also performed including linearity, efficiency, sensitivity, repeatability and its application to commercial samples. The designed primer (CYTBCA3-kh) consisted of F: CCT TAG CCA ATG CCT ATT C and R: GCG ACT TGT CCG ATA ATG. The results showed that CYTBCA3-kh primer was specific to DNA extracted from CM and that extracted from meatballs containing CM using optimized annealing temperature of 50.6 o C. The detection limit reported was 50 pg DNA corresponding to 0.1% CM in meatballs containing CM and beef. The relative standard deviation (RSD) for precision assay met the required acceptance criteria. The validated real-time PCR using CYTBCA3-kh primer could successfully identify CM in meatball formulation for halal authentication analysis. The developed method was potential to be developed as official standard method for CM detection in meatball products.