12S rRNA mitochondrial gene as marker to trace Sicilian mono-species dairy products

For a rapid, specific and sensitive identification of cows', ewes' and goats' milk in mono-species Sicilian dairy products, species-specific duplex-PCR protocol was applied. DNA samples from blood and experimental cheeses of Sicilian autochthonous breeds were extracted to amplify the 12S rRNA (and part of 16S rRNA in case of Ovis aries) mitochondrial species-specific gene fragment. The use of species-specific primers for Bos taurus, Capra hircus and Ovis aries species, after electrophoresis on agarose gel, yielded fragments of 256 bp, 326 bp and 172 bp, respectively. Amplification by duplex-PCR of DNA pools from two species showed detection thresholds of 0.1% of “contaminant” DNA in each mixture. Finally, duplex-PCR assay was applied to experimental cheeses in order to detect the minimum threshold of DNA belonging to one species in cheese made with milk of two species. The results showed a sensitive threshold of 0.1% of ewes' milk in cows' and goats' cheeses, 0.1% of cows' milk in ewes' and goats' cheeses, and finally 0.1% of goats' milk in cows' and ewes' cheeses. The proposed assay represents a rapid and straightforward method of species traceability for the detections of adulteration in Sicilian mono-species dairy products.