Establishment and Application of Multiplex Real-Time PCR Method for Rapid Detection of Four Animal-Derived Components in Donkey Meat Products

A multiplex real-time polymerase chain reaction (PCR) method for simultaneous rapid detection of donkey-, horse-, pig- and duck-derived components was established. For this purpose, the Nad5, ATpase6, ATP8 and cytb were used as target genes to design specific primers and TaqMan probes, and the 18S rRNA gene was used as internal reference gene. The developed method was validated and applied to detect simulated adulterated meat products, simulated processed meat products and actual donkey meat samples. This method was characterized by high-throughput, and excellent sensitivity and specificity. The system had no cross-reaction to non-target sources when the cycle threshold (Ct) value did not exceed 35.0. The sensitivity was 2 × 10^-4 ng/μL of template DNA. The detection limit for raw and cooked meat was 0.001% and 0.01% of the meat content, respectively. The results of this method for 100 actual samples were consistent with those of the national standard method, indicating that the multiplex real-time PCR method can be used for the detection of commonly adulterants in meat and meat products.