A multiplex real-time PCR approach for identification and quantification of sheep/goat, fox and murine fractions in meats using nuclear DNA sequences

Abstract Adulterating undeclared animal components into meats may cause health problems. In this study, nuclear DNA sequences for specific detection of sheep/goat, fox and murine (mouse, rat and hamster) were selected to design primers and probes for determination of mutton adulteration. The inter-specific specificity and intra-specific conservation were strictly evaluated in 19 different animal species and breeds, respectively. These three nuclear DNA sequences were confirmed to be fixed copy sequences by relative quantitative PCR. Furthermore, a multiplex qualitative and quantitative PCR system with sensitivity of 0.05 ng was developed to efficiently discriminate sheep/goat (237 bp), fox (211 bp) and murine (160 bp) in one reaction. Finally, several mixed DNA samples of goat, fox and rat were quantitatively analyzed by multiplex real-time PCR method, showing good accuracy (R.E. from 1.64% to 18.43%) and precision (R.S.D. from 0.51% to 12.70%). The method developed in this study will provide a powerful technology to combat meat adulteration and protect consumer health.