Multiplex and real-time PCR for qualitative and quantitative donkey meat adulteration

Donkey meat is a rich source of protein excellent in indispensable amino acid and low in fat. Its quality has received great attention due to its high nutritional and medicinal value to human beings. Due to donkey resource scarcity and gradually increasing market demand for donkey meat products, adulterated donkey meat products with other low cost animal meat, especially sheep, cattle, pig and horse, were often found in market and had raised widespread concern in recent years. In this study, total 300 mg samples of binary mixtures, containing 90%, 50%, 25%, 10%, 5%, 1% (w/w) of donkey in horse meat respectively, were used for DNA extraction. Both conventional multiplex and real-time PCR techniques were developed for qualitatively and quantitatively detecting the adulteration in donkey meat. The multiplex PCR allowed rapid and simultaneous qualitative detection of donkey and four common adulterated species including sheep, cattle, pig and horse. Moreover, a normalized real-time PCR assay was further developed to detect and quantify the donkey mitochondrial DNA, and the results of simulate adulteration suggested this method could quantitatively detect donkey meat in range of 1–90% with high trueness. The results illustrated that both methods might be applied to detect commercial foodstuff adulteration. Therefore, both improved conventional multiplex and real-time PCR methods were developed to achieve qualitative and quantitative detection of the adulteration in donkey meat products.