Application of family-specific primers in multiplex real-time PCR for meat categories screening

Abstract

Meat adulteration has been a quality issue in entire meat supply chain, which can harm consumers’ economic and health interests seriously. In order to prevent adulteration of mutton and reduce the cost of testing, three family-specific primers were designed and then a triplex real-time PCR method with melting curve analysis was developed, which could identify three meat categories simultaneously. The primers were designed from mitochondrial gene including 16S rRNA and 12S rRNA. The primer CMRC-Ovis/Capra3 can only recognize Ovis category including sheep and goat without recognizing other species. Similarly, primer CMRC-Canidae2 can only identify Canidae category including dog, fox and racoon-dog; and CMRC-Poultry3 can only identify poultry category including chicken, duck and goose respectively. The amplicons’ melting temperature of three categories were 72.45∼75.91 °C, 77.25∼78.76 °C and 84.58∼87.40 °C, respectively. The specificity, universality, detection limit and practicability were validated and the results showed that all family-specific primers have good specificity and universality. This method has low detection limit (0.001∼0.1 ng DNA), strong reproducibility, and good practicability, which indicated a good application prospect in the screening of adulterated species in mutton products.