A rapid and reliable multiplex PCR assay for simultaneous detection of fourteen animal species in two tubes

A simple and rapid method for animal species identification to prevent food adulteration based on mitochondrial DNA using two independent multiplex polymerase chain reactions (PCRs) and microchip electrophoresis was developed. This method was designed to identify fourteen domestic animals (Group I: cattle, donkey, dog, fox, raccoon-dog, deer and horse; Group II: pig, sheep, goat, chicken, duck, cat and mouse) simultaneously using ten pairs of primers and three of which were degenerate primers. Sequences for species-specific primers were generated based on mitochondrial genes, including 12S rRNA, 16S rRNA, ND2 and CO I. This method was validated in terms of the specificity, sensitivity and practicability, and the developed multiplex PCR method was able to correctly identify animal species of raw meats and processed meat products. The detection limits of two multiplex PCRs were 0.02 ng DNA for animal species in Group I and 0.2 ng DNA for Group II, respectively.