A novel closed-tube method based on high resolution melting (HRM) analysis for authenticity testing and quantitative detection in Greek PDO Feta cheese

Animal species identification of milk and dairy products has received increasing attention concerning food composition, traceability, allergic pathologies and accurate consumer information. Here we sought to develop an easy to use and robust method for species identification in cheese with emphasis on an authenticity control of PDO Feta cheese products. We used specific mitochondrial DNA regions coupled with high resolution melting (HRM) a closed-tube method allowing us to detect bovine, ovine and caprine species and authenticate Greek PDO Feta cheese. The primers successfully amplified DNA isolated from milk and cheese and showed a high degree of specificity. HRM was proven capable of accurately identifying the presence of bovine milk (not allowed in Feta) down to 0.1% and also of quantifying the ratio of sheep to goat milk mixture in different Feta cheese commercial products. In conclusion, HRM analysis can be a faster, with higher resolution and a more cost effective alternative method to authenticate milk and dairy products including PDO Feta cheese and to quantitatively detect its sheep milk adulterations.