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Koumiss is a popular dairy product in many lands, traditionally prepared from mare milk with spontaneous fermentation. Mare milk and its fermented derivates are more expensive than cow milk and its fermented derivates, and the possibility exists for producers and dealers to adulterate equine products with bovine items. In this work, we described the development of a triplex real-time PCR based on species-specific TaqMan probes for identification of bovine and equine DNA in milks and dairy products. In addition, a novel designed endogenous control was simultaneously amplified to eliminate possible false negatives. With this methodology, bovine and equine DNA were specifically identified by employing developed primers and probes. The limits of detection of this method were 0.001 ng for cow milk, yogurt, and mare milk, and 0.005 ng for sour soup and koumiss, respectively. In addition, the triplex real-time PCR assay for authentication of animal-derived products was effectively validated using binary DNA and milk mixtures, exhibiting well in terms of specificity, sensitivity, and reproducibility. In short, the triplex PCR assay was verified to be a time-saving and money-saving technique for the identification of bovine and equine DNA in milks and dairy products.

Authentication of dairy and meat products is important to ensure fair competition, consumer benefit, and food safety. The large difference in price between camel and cow milk may be an incentive to adulterate camel dairy products with cow-derived foodstuffs. However, no studies so far have used triplex real-time PCR with an endogenous control to identify camel and cow origins in dairy and meat products. In this study, we developed a triplex real-time PCR assay based on amplification of mitochondrial 12S ribosomal DNA for the authentication of camel-derived dairy and meat products. This method was applied to identify camel and cow DNA in milk, yogurt, cheese, milk powder, milk beverage, meat products, and mixtures with milk and meat. Concentrations as low as 1 to 5% and 0.1% camel milk and meat, respectively, were detected in the mixtures, and 1 to 5% and 0.1% cow milk and meat, respectively, were identified via this approach. The limits of detection were 0.005 to 0.0025 ng, 0.05 to 0.001 ng, 0.001 to 0.0005 ng, and 0.00025 to 0.0001 ng of DNA in camel milk, camel yogurt, commercial camel milk beverage, and camel meat, and from 0.0025 to 0.001 ng, 0.5 to 0.001 ng, 1 to 0.05 ng, 0.01 ng, 0.001 ng, 0.0005 to 0.00025 ng, 0.0005 to 0.00025 ng, and 0.005 ng of DNA from cow milk, yogurt, cheese, acidic whey, milk powder, beef, beef jerky, and beef sausage, respectively. Different dairy and meat samples of camel and cow origins had a range of authentication limits and limits of detection. The designed triplex real-time PCR assay was shown to be a specific, sensitive, and efficient technique for the identification of camel and cow DNA in foodstuffs.

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