A Novel Universal Primer Multiplex Real-Time PCR (UP-M-rtPCR) Approach for Specific Identification and Quantitation...

Adulteration of meat with carnivorous animals (such as cats, dogs, foxes, and minks) can cause ethical problems and lead to disease transmission; however, DNA quantitative methods for four carnivorous species in one tube reaction are still rare. In this study, a carnivore-specific nuclear DNA sequence that is conserved in carnivorous animals but has base differences within the sequence was used to design universal primers for its conserved region and corresponding species-specific probes for the hypervariable region. A novel universal primer multiplex real-time PCR (UP-M-rtPCR) approach was developed for the specific identification and quantitation of cat, dog, fox, and mink fractions in a single reaction, with a 0.05 ng absolute limit of detection (LOD) and 0.05% relative LOD. This approach simplifies the PCR system and improves the efficiency of simultaneous identification of multiple animal-derived ingredients in meat. UP-M-rtPCR showed good accuracy (0.48-7.04% relative deviation) and precision (1.42-13.78% relative standard deviation) for quantitative analysis of cat, dog, fox, and mink DNA as well as excellent applicability for the evaluation of meat samples.