Collaborative ring trial for the detection of cow, sheep and goat milk in dairy samples by real-time PCR targeting mitochondrial DNA

Authentication of dairy product composition is vital for consumer protection and food safety. This study validated 4 real-time PCR methods targeting mitochondrial DNA from cattle, sheep, goat, and total mammals in a collaborative ring trial. Twelve well-characterised dairy samples – including milk, yoghurt, Picodon-type cheese, and Feta-type cheese ‒ were produced from sheep and goat milk with 1–33.3% cow milk admixture for use in a ring trial involving 15 laboratories. Additional samples from various production stages increased the total to 50, which were also analysed by a single laboratory. All 4 real-time PCR assays demonstrated high specificity and sensitivity, with no false positives or negatives observed in the collaborative trial. Quantitative analysis of species proportions showed good precision across laboratories. However, the proportion of cattle DNA was consistently underestimated due to lower somatic cell counts in cow milk. This bias was effectively reduced by calibrating with milk-based reference samples rather than DNA standards. The methods performed reliably across diverse dairy matrices and production stages. Duplex PCR formats matched the performance of singleplex assays while reducing both time and reagent use. Quantitative results support differentiation between economically motivated adulteration and incidental contamination, although precise determination of cow milk admixture remains challenging due to biological variability. The validated real-time PCR methods described in here are included in the Official Collection of Methods of Analysis (ASU), published by the German Federal Office of Consumer Protection and Food Safety (BVL).