Holstein's milk detection in cheeses inferred from melanocortin receptor 1 (MC1R) gene polymorphism.

For some French Registered Designation of Origin (RDO) cheeses Prim'Holstein's milk is not allowed for cheese making (e.g., Reblochon, Abondance, and Beaufort cheeses). To find molecular markers for Prim'Holstein's milk detection in RDO cheese, four genes affecting coat color in cattle (c-kit, MGH, TYRP1, and MC1R) have been sequenced for three mountain breeds and the Prim'Holstein breed. Only the MC1R gene (E-locus) has shown variation between the four breeds. Among the 25 French and Italian breeds sequenced for the MC1R gene, only the Vosgienne breed has presented the same allele as the black Prim'Holstein breed (E(D)). A quick and easy DNA-based method to detect Holstein's milk in RDO cheese is proposed based on ED allele detection. A DNA extraction from cheese, a preamplification of the gene and a competitive oligonuleotide priming PCR on MC1R mutations were performed. Using an automated sequencer, differences in fluorescence and fragment size reveal the allele type. This simple approach provides good reproducibility and is shown to be relatively sensitive, with a detection limit of about 1% of Holstein's milk in milk curd.