Droplet Digital PCR (ddPCR) Analysis for the Detection and Quantification of Cow DNA in Buffalo Mozzarella Cheese

SIMPLE SUMMARY: Buffalo mozzarella cheese, sold as a Protected Designation of Origin (PDO) product, is made exclusively from Mediterranean buffalo (Bubalus bubalis) milk. To maximize their profits and overcome seasonal shortages of buffalo milk, some producers have started to produce “PDO” buffalo mozzarella cheese by adulterating buffalo milk with milk from different species. such as bovine, thus defrauding consumers. This practice has led the Italian government to reinforce controls on food mislabeling and fraud through traceability mechanisms. The aim of this work is the development of a molecular assay—droplet digital Polymerase Chain Reaction technique (ddPCR)—able to detect the DNA of cow and/or buffalo milk in PDO buffalo mozzarella cheese, thus revealing fraud. The results have highlighted that, thanks to its high precision and sensitivity, the ddPCR could represent an efficacious means of supporting the official controls aimed at combating the adulteration of buffalo mozzarella cheese with bovine milk. ABSTRACT: Buffalo mozzarella cheese is one of the most appreciated traditional Italian products and it is certified as a Protected Designation of Origin (PDO) product under the European Commission Regulation No. 1151/2012. It is obtained exclusively from buffalo milk. If made from cow milk, or a mixture of buffalo and cow milk, buffalo mozzarella cheese does not qualify as a PDO product. In order to maximize their profits, some producers market buffalo mozzarella that also contains cow milk as a PDO product, thus defrauding consumers. New methods for revealing this fraud are therefore needed. One such method is the droplet digital Polymerase Chain Reaction (ddPCR). Thanks to its high precision and sensitivity, the ddPCR could prove an efficacious means for detecting the presence of cow milk in buffalo mozzarella cheese that is marketed as a PDO product. ddPCR has proved able to detect the DNA of cow and/or buffalo milk in 33 buffalo mozzarella cheeses labelled as PDO products, and experimental evidence could support its application in routine analyses.