Establishment and Application of Multiplex PCR Scheme for Simultaneously Distinguishing Muscle Tissues of Mink, Fox, and Raccoon Dog from Conventional Livestock and Poultry

It is possible and risky for fur animal carcasses to be mixed into meat products, which is a potential danger for meat quality safety and human health. Therefore, meat validation of quality and constituents is crucial. A variety of methods have been developed to identify muscle tissues of different species. However, most of these methods have the disadvantages of poor repeatability, complex operation and low efficiency, and cannot simultaneously detect multiple species of muscle tissue. The purpose of this study was to construct a multiplex PCR protocol to detect the samples of mink, fox, and raccoon dog. In this study, the specific primers of mink, fox, and raccoon dog were designed according to the variable region sequence of mitochondrial cytochrome b ( Cytb ) gene. The primers showed good specificity and 50 ℃ was determined as the optimal annealing temperature. The lowest concentration of DNA template of mink, fox, or raccoon dog that could be determined simultaneously by a single tube was 1 pg/µL. Clinical tissue samples detect analysis test results showed that this method could identify whether the tissue samples of three fur animals were mixed from the muscles of chickens, ducks, dogs, cattle, sheep, pigs and rabbits in one PCR reaction simultaneously. In conclusion, the scheme exhibited the advantages of convenient operation, low cost, strong species specificity, high sensitivity, good stability, and repeatability. The systematic optimized inspection process can be applied to meat detection to ensure veterinary public health safety, which has important scientific significance, production, public health, and safety significance.