A sensitive DNA-based fluorometric method for milk authenticity of dairy products based on spectrally distinct microspheres

Milk authenticity of dairy products is of great demand in order to protect consumers from fraudulent products and health risk. Milk-based products produced by goat and sheep milk are located at the top of the list of healthy dairy products, earning the increasing interest of consumers. As a result, these products are extremely vulnerable to adulteration with milk of lower added and nutritional value for profit. In addition, adulterant milks, such as bovine milk, have been accused to cause allergic reactions, especially to sensitive human groups. For these reasons, there is a great need for sensitive and robust analytical methods for milk adulteration detection. This work focused on the development of a DNA-based analytical method based on hybridization assay on spectrally distinct microspheres for adulteration detection of goat and sheep dairy products with bovine milk. The method includes (a) DNA isolation from dairy products, (b) amplification of DNA sequences specific to each animal species and (c) hybridization assay on the surface of distinct microspheres. The identification of the animal origin is accomplished by species-specific oligonucleotide probes coupled to the microspheres and the hybrids formed are detected by a streptavidin-phycoerythrin conjugate. The microspheres are finally analyzed by flow cytometry. The fluorescence emitted by the reporter molecule phycoerythrin is proportional to the concentration of the DNA content in the sample. As low as 0.01% bovine milk in goat milk and 0.05% in sheep milk was able to be detected by the proposed method. The method offers very good detectability, sensitivity and reproducibility.