Heptaplex Polymerase Chain Reaction Assay for the Simultaneous Detection of Beef, Buffalo, Chicken, Cat, Dog, Pork, and Fish in Raw and Heat-Treated Food Products

Species authentication of meat and fish products is crucial to safeguard public health, economic investment, and religious sanctity. We developed a heptaplex polymerase chain reaction assay targeting short amplicon length (73–198 bp) for the simultaneous detection and differentiation of cow, buffalo, chicken, cat, dog, pig, and fish species in raw and processed food using species-specific primers targeting mitochondrial cytb, ND5, and 16s rRNA genes. Assay validation of adulterated and various heat-treated meatball matrices showed excellent stability and sensitivity under all processing conditions. The detection limit was 0.01–0.001 ng of DNA under pure states and 0.5% meat in meatball products. Buffalo was detected in 86.7% (13 out of 15) of tested commercial beef products, while chicken, pork, and fish products were found to be pure. The developed assay was efficient enough to detect target species simultaneously, even in highly degraded and processed food products at reduced time.