Abstract: 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.

Key words: mitochondrial gene; nuclear gene; horse ingredient; donkey ingredient; mule ingredient; sequencing analysis