37| VOLUME 79 ISSUE 1 | MAY A Molecular Procedure for Detecting Dairy Products Adulteration Beatrice Ana-Maria TUDOR, Oana-Maria BOLDURA * , Călin MIRCU, Camelia TULCAN, Simona MARC, Bianca Cornelia LUNGU, Iuliu TORDA, Ioan HUŢU Faculty of Veterinary Medicine, Banat’s University of Agricultural Sciences and Veterinary Medicine” King Michael I of Romania” from Timisoara, Romania *Corresponding author: O. Boldura e-mail: oanaboldura@usab-tm.ro RESEARCH ARTICLE Abstract Dairy products are an essential component of human nutrition and therefore are prone to adulteration. Although many methods have been developed based on the analysis of protein substances by which adulteration is detected, they have low efficiency in the case of processed products. In this context, methods based on DNA analysis have become the best choice, although the results are not universally valid and must be adapted to the composition and local specificities. Here is presented a study of the applicability of a fast and cost-effective DNA- based method to determine the species composition of dairy products by performing a qualitative triplex PCR detection technique in the analysis of local dairy products. The procedure is adapted to the domestic market and considers experimental models developed and well defined internationally. In the in-house validation of this method, three species of ruminants were considered: beef, sheep, and goat. The reference material was represented by cheeses prepared in our laboratory in strictly controlled percentage mixtures. Total genomic DNA isolated from the reference cheeses were used to develop a triplex PCR method, which involves the simultaneous detection of the three species studied using a mixture of three pairs of primers. The reference materials proved to be optimal for such studies and have been used successfully in the process of validating the method of detecting the composition of commercial cheeses. Keywords: dairy products, adulteration, multiplex PCR, mitochondrial DNA. INTRODUCTION Milk and dairy products are an essential component of human nutrition and are most commonly consumed by children and the elderly. Annually, out of the total milk produced globally, 85% is cow's milk, 11% buffalo's milk, and only 2% sheep's milk and goat's milk. A large part of this milk is processed into dairy products, about 60% (Sala et al., 2015). It is considered that 30% of the need for lipids and proteins and 80% of the daily calcium requirement is provided by dairy products. Fraud by substituting sheep's, goats, or buffalo's milk with cow's milk has become common. Milk was the second most adulterated food reported in the scientific record by 14% (Moore et al., 2012). Therefore, various detection methods such as immunological, electrophoretic, chromatographic, and DNA based have been developed to detect adulterations. The most used techniques for detecting adulteration of dairy products by replacing milk from different species are various immunological analyses developed over the years, including enzyme-linked immunosorbent assays (Hurley et al., 2004). However, protein- based methods for species identification may fail after excessive proteolysis or heat-induced denaturation of indicator proteins. Therefore, it is suggested that genomic DNA from somatic cells in milk persists in matured cheese and can be amplified and analyzed for species identification. Currently, in the European Community, isoelectric focusing of γ-casein after plasmolysis is the reference Received: 26 September 2021 Accepted: 03 March 2022 Published: 15 May 2022 DOI: 10.15835/buasvmcn-vm:2021.0034 © 2022 Authors. The papers published in this journal are licensed under the Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International License