A TaqMan real-time polymerase chain reaction (PCR) assay was developed for the simultaneous detection of chicken-, duck-, swine- and bovine-derived ingredients in quick-frozen food. Specific primers and TaqMan probes were designed and synthesized using the transforming growth factor beta-3 (TGFB3) gene of chicken, the prion protein (PRNP) gene of swine and the growth hormone (GH) gene of duck and bovine as target genes. The PCR reaction systems and conditions were optimized. The developed real-time PCR assay demonstrated good specificity, in which specific amplifications were observed for chicken, duck, swine and bovine. The assay also showed high sensitivity with limits of detection for chicken-, duck-, swine- and bovine-derived ingredients of 10.0, 1.0, 10.0 and 10.0 pg/μL, respectively. The PCR assay could detect the four kinds of animal-derived ingredient in 34 commercial quick-frozen foods. Further analysis revealed that the results of the PCR assay for 15 quick-frozen foods were inconsistent with the ingredients on the label. The real-time PCR assay developed in this study has good specificity and sensitivity, and it is simple to operate and suitable for the rapid detection of animal-derived ingredients in quick-frozen foods.

Both horse- and donkey-derived ingredients have been detected in mule meat by real-time polymerase chain reaction (PCR) as described in China’s industry standards for detection of horse (SN/T 3730.5-2013) and donkey (SN/T 3730.4-2013) ingredients in food and feed, respectively. This contradicts the theory of strict maternal inheritance of mitochondrial DNA (mtDNA). Therefore, in this study, 3 horse meat samples, 3 donkey meat samples and 3 mule meat samples were detected by mitochondrial gene and nuclear gene sequencing based on PCR and the China’s industry standard methods for horse and donkey ingredients, respectively, and the results of the SN/T 3730.4-2013 method for mule meat were analyzed. According to the results of mitochondrial gene and nuclear gene sequencing, all 3 mule meat samples were derived from mules. Both donkey and horse ingredients were detected in the 3 mule meat samples by the SN/T 3730.4-2013 and SN/T 3730.5-2013 methods. The cycle threshold (Ct) of the SN/T 3730.5-2013 method for horse ingredient was in the range of ≤ 20.00, and that of the SN/T 3730.4-2013 method for donkey ingredient were in the range of 25.00-35.00. The sequencing results of PCR products using the primers described in the SN/T 3730.4-2013 method showed that the 3 mule meat samples had no homology with horse or donkey meat. This might be because the SN/T 3730.4-2013 target sequence appeared in the form of nuclear mitochondrial DNA segments in low repeat numbers in the mule nuclear genome, and some base insertions and deletions occurred. The possibility that mule ingredient may be present should be considered when the Ct value of the SN/T 3730.4-2013 is ≤ 20.00, while the Ct value of the SN/T 3730.5-2013 is in the range of 25.00-35.00 for horse and donkey ingredients in known samples of single animal-derived ingredients, respectively.