A multiplex PCR method for detection of five animal species in processed meat products using novel species-specific nuclear DNA sequences

The continuous development of fast and simple new methods to identify animal-derived ingredients is very important for the authentication of meat products. This study intended to develop a multiplex PCR method using new species-specific nuclear DNA (nDNA) sequences for the detection of ingredients derived from sheep/goat, bovine, chicken, duck and pig in meat products. Sequence alignment analysis in 53 species showed high specificity of species-specific nDNA. Species-specific primers were designed on the conservative region of each species-specific nDNA sequence. The specificity and conservation of the sequences and primers were verified by PCR reaction and sequencing with the limit of detection down to 0.5 ng. Then, a species-specific multiplex PCR method was developed and optimized to simultaneously detect sheep/goat (237 bp), bovine (223 bp), chicken (192 bp), duck (168 bp) and pig (154 bp) in one reaction. Various processed meat products containing one or more animal-derived ingredients were detected by the developed multiplex PCR method, and the results were consistent with their labeled meat species. Our study provides a fast and simple detection method for regulating labeling of animal-derived ingredients in meat products.