Use of duplex polymerase chain reaction (Duplex-PCR) technique for the identification of bovine and water buffalo milk used for making mozzarella cheese

Molecular biology technique have been used for species identification in food of animal origin in relatively recent years. A polymerase chain reaction (PCR) based method, the multiplex PCR, was recently applied to species identification in meat and meat products. It allow co-amplification of separate regions of a single gene or specific fragment, each typical of a different animal species in a single PCR reaction, using different pair of primers in the same reaction mix. In the present paper, the duplex-PCR technique is proposed to identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk). Because of its lower cost, undeclared bovine milk is added to water buffalo milk for making different kind of mozzarella cheese. The results of this experiment indicate the applicability of this method, wich showed an absolute specificity for the two species and a high sensitivity even down to low DNA concentration (1pg). In bovine and water buffalo mixtures of both milk and mozzarella cheese, the minimum concentration tested was 1% of bovine in water buffalo milk and water buffalo in bovine milk. The importance of the somatic cell content in raw milk is also discussed with special reference to the evaluation of mixtures (milk and cheese) of the two species.