Application of Industrial Standard Methods for Detection of Horse- and Donkey-Derived Ingredients for Detecting Mule Meat

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

Rapid detection of duck ingredient in adulterated foods by isothermal recombinase polymerase amplification assays

Duck is often used in meat fraud as a substitute for more expensive meats. Rapid detection of duck ingredient in meat products is of great significance for combating meat fraud and safeguarding the interests of consumers. Therefore, we aim to develop duck-specific recombinase polymerase amplification (RPA)-based assays for the rapid detection of duck ingredient in animal-derived foods. Using Cytb gene as target, the real-time RPA and RPA combined with lateral flow strips (LFS RPA) were developed successfully for the rapid detection of ducks in 20 min at 39 °C and 40 °C, respectively. The assays did not show cross-reactions with 6 other livestock and poultry. The developed RPA assays could detect 10 pg duck genomic DNA per reaction and 0.1 % (w/w) duck ingredient in duck and mutton mixed powder within 30 min, including a rapid nucleic acid extraction. Furthermore, duck ingredient could be detected in 30 different actual foods including heat-processed meats and blood products. Therefore, duck-specific real-time RPA and LFS RPA assays were successfully developed with good specificity and sensitivity, which could enable rapid detection of duck ingredient in the field and provide technical support for combating the meat fraud