High resolution melting is a useful tool to detect animal species sources of various milk types

Accurate identification of animal species sources in milk have become quite important due to adulteration of high-priced milk types in the dairy industry. To date, milk identification methods have mainly depended on biochemical properties or physical properties detected by spectroscopic methods. The current study aimed to develop an easy to use and sensitive DNA-based High resolution melting (HRM) assay to identify animal species and detect cross-adulteration of water buffalo, bovine, goat, sheep, camel and donkey milks. HRM compatible designed primer set, targeted mitochondrial region, successfully amplified the specific targeted region for six animal species DNA and showed a high degree of specificity based on nucleotide variations. Capillary electrophoresis analysis validated the specific amplicons and determined the amplicon lengths as 114 bp for bovine, goat, sheep, and camel, 115 bp for water buffalo, and 121 bp for donkey. HRM analysis showed a clear discrimination for water buffalo-bovine, camel-bovine and donkey-bovine adulteration down to 0.5%, and goat-sheep adulteration down to 1% in the milk admixtures. The efficacy of the method was also confirmed by its standard curve with a very high correlation coefficient In conclusion, the designed HRM assay allows for the rapid, sensitive and cost-effective authentication of milk and dairy products.

The online version contains supplementary material available at 10.1007/s13197-023-05705-3.