The detection of horse and donkey using real-time PCR

We have developed real-time PCR assays specific for horse and donkey, applicable to the detection of low levels of horse or donkey meat in commercial products. Primers, designed to the mitochondrial cytochrome b gene, were 3′ mismatched to closely related and other commercial species. Amplification of non-target species DNA was prevented by truncation of primers at the 5′ position, thereby conferring complete specificity. Both assays were highly sensitive and detected the presence of 1 pg of donkey template DNA or 25 pg of horse template DNA when assessed using dilutions of DNA in water. Model food samples, spiked with horse or donkey muscle and commercial products containing horse, were successfully tested for the presence of horse or donkey, demonstrating the applicability of the assays to food products.