A duplex polymerase chain reaction (PCR) was developed to identify the milk of bovine and buffalo species in cheese products, particularly in mozzarella cheese, a typical Italian cheese made from buffalo's milk. Two sets of primers were designed on the basis of the alignment of the sequence codifying mitochondrial cyt b available in the GenBank database. The primers proved to be species-specific, giving rise to 279-bp (bovine) and 192-bp (buffalo) amplified fragments. Since the amplification conditions for bovine and buffalo primers were identical, a duplex PCR was successfully applied to identify the two species in a single reaction step. This technique, when used to test cheese products from the retail trade, allowed the detection of partial or even total substitution of cow's milk for buffalo's milk, in some cases in samples of cheese misleadingly labeled “pure buffalo” mozzarella.

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