Development of multiplex-PCR protocol to amplify 12S and 16S rRNA genes of mtDNA for traceability of Sicilian mono-species dairy products.

Species identification in dairy sector is important not only to safeguard public health but also to verify compliance with the Production Regulations of many typical dairy products (PDO/PGI). The most common fraud in dairy sector is represented by the mixture of milks from different species, resulting in mislabeling of protected designation of origin (PDO) products. For a rapid, specific and sensitive identification of cattle’s, sheep’s and goat’s milk in mono-species Sicilian dairy products, species-specific multiplex-PCR protocol was developed. DNA from blood and experimental cheeses of Sicilian autochthonous breeds was used to amplify the 12S and 16S rRNA genes of the mitochondrial DNA. Modicana and Cinisara cattle breeds; Comisana, Pinzirita, and Valle del Belice sheep breeds; Girgentana, Maltese and Derivata di Siria goat breeds were the sampled autochthonous breeds. The use of species-specific primers for bovine, caprine and ovine species, allowed amplifying of fragments with different lengths, 256 bp, 326 bp, and, 172 bp, respectively. In the next step, amplification by multiplex-PCR of pools containing mixtures of DNA from two species at different percentage, showed the following sensitive thresholds: 0.1% for bovine/caprine and ovine/caprine DNA mixtures and 0.5% for bovine/ovine and ovine/bovine DNA mixtures. Finally, multiplex-PCR assay was applied to bovine/ovine and ovine/bovine experimental cheeses to detect the minimum thresholds for both species. The results showed the sensitive thresholds of 0.1% for bovine/ovine cheeses and 0.5% for ovine/bovine ones. The proposed assay represents a rapid and straightforward method for the detections of adulteration in Sicilian mono-species dairy products. Therefore, the ability to detect low levels of contaminating milk could be interesting to safeguard not only monospecies dairy products protected by European labels but also allergic or intolerant subjects.