Development of a real-time PCR assay for the detection of cow and donkey milk

A real-time PCR allelic discrimination TaqMan assay based on the analysis of a single nucleotide polymorphism enabling the differentiation of cow (Bos taurus) and donkey (Equus asinus) milk was developed. Specific primers and probes were designed on the mitochondrial cytochrome c oxidase subunit I gene. The primers were designed upstream and downstream the chosen diagnosis site in a conserved region. Two probes were designed to specifically hybridise to B. taurus and E. asinus sequences. The test allowed the discrimination of bovine and donkey DNA in all blood and pure milk samples giving an unambiguous result plot of rapid and easy interpretation. The detection threshold was 2 % of cow milk in donkey milk. The applicability of the method to matrices containing degraded DNA was demonstrated by analysing samples of raw donkey and cow milk autoclave-treated (121 °C for 15 min). Finally, the assay when applied to milk samples collected from the retail trade has confirmed the species indicated in the label. Furthermore, the assay represents a potentially valuable diagnostic tool for species identification in dairy products for allergic people.