Optimization of Molecular Genetic Method for Identification of Dairy Raw Materials

Abstract

Introduction: This article addresses the pressing issue of ensuring the quality and safety of dairy products by combating adulteration, a challenge that continues to plague the dairy industry. Adulteration, often driven by cost-cutting motives, involves altering the composition, quality, or origin of dairy products, even in the face of tightened control measures and improved monitoring systems. Specifically, this study hones in on the problem of species-specific adulteration, particularly the substitution of goat milk with cow milk due to the significant cost differential. Detecting and identifying such adulteration is crucial, and while traditional methods like electrophoresis and chromatography have been used, they are often expensive and labor-intensive. The article explores the use of molecular genetic methods, particularly polymerase chain reaction (PCR), as a more efficient and accurate means of identifying species-specific milk adulteration.  Methods like PCR offer high specificity, sensitivity, speed, and the ability to perform quantitative and multiplex analyses.Purpose: The objective of this study is to optimise a method for species identification of dairy products by PCR-based screening using DNA isolated from cow’s and goat’s milk comatic cells.Materials and Methods: This research conducted at the Central Laboratory of Microbiology, All-Russian Research Institute of Dairy Industry, focuses on milk species identification of cattle and small ruminants using a specific set of primers targeting the polymorphic amylogenin gene. The study involves PCR screening with DNA extracted from cow’s and goat’s raw milk utilizing a MiniAmp instrument for the analysis. Qualitative and quantitative assessments of DNA preparations were performed, measuring DNA concentration with a Qubit 4 fluorimeter and Qubit dsDNA BR Assay Kit. PCR analysis was carried out in 25 μl reactions, including key components such as 5xScreen Mix, specific primers, DNA samples, and mQ H2O. The amplification program comprised denaturation, annealing, and elongation steps over a series of cycles.Results: The experiment was aimed at assessing the suitability of primers SE47 and SE48 for amplification of nuclear DNA of milk somatic cells of cattle (Bos taurus) and goats (Capra hircus). Initially, a calculated annealing temperature of 56 °C was used in the PCR reaction, yielding in numerous nonspecific fragments appeared on the electropherogram. To solve this problem, we gradually increased the annealing temperature, which resulted in a significant decrease in the nonspecific fragments number and their complete absence at annealing temperature of 70°C.Conclusion: We succeeded in optimizing a PCR-based detection system for the milk species identification of cattle and small ruminants. The results obtained confirm the possibility of using genomic DNA of milk somatic cells for the successful amplification of species-specific nuclear markers, but there remains a need for further research to determine the sensitivity of the PCR system and the possibility of its use in the analysis of milk processing products.