Simultaneous detection of cow and buffalo milk in mozzarella cheese by Real-Time PCR assay

A Real-Time PCR Allelic Discrimination TaqMan assay based on the analysis of one diagnosis position enabling the identification of cows’ and buffalo milk in dairy products was developed. Specific primers and probes were designed on the mitochondrial cytochrome b gene. In particular, primers were designed upstream and downstream the chosen diagnosis site in a well conserved region for both Bos taurus and Bubalus bubalis. Two probes were designed to specifically hybridise to B. taurus and B. bubalis sequences. This technique proved to be species-specific when tested on blood and pure milk samples giving an unambiguous result plot of rapid interpretation. The detection threshold, evaluated by means of different dilutions, was 2% of cow milk in buffalo milk. The test, when applied to cheese samples from the retail trade, enabled the identification of partial or total substitution of cow for buffalo milk. Furthermore, it is relatively rapid, as both species can be detected in one step, with no need for any post-PCR manipulation.