Development of a real-time PCR assay for the detection of cow and donkey milk

Abstract

A real-time PCR allelic discrimination TaqMan assay based on the analysis of a single nucleotide polymorphism enabling the differentiation of cow (Bos taurus) and donkey (Equus asinus) milk was developed. Specific primers and probes were designed on the mitochondrial cytochrome c oxidase subunit I gene. The primers were designed upstream and downstream the chosen diagnosis site in a conserved region. Two probes were designed to specifically hybridise to B. taurus and E. asinus sequences. The test allowed the discrimination of bovine and donkey DNA in all blood and pure milk samples giving an unambiguous result plot of rapid and easy interpretation. The detection threshold was 2 % of cow milk in donkey milk. The applicability of the method to matrices containing degraded DNA was demonstrated by analysing samples of raw donkey and cow milk autoclave-treated (121°C for 15 min). Finally, the assay when applied to milk samples collected from the retail trade has confirmed the species indicated in the label. Furthermore, the assay represents a potentially valuable diagnostic tool for species identification in dairy products for allergic people.

Validation of a fast real-time PCR method to detect fraud and mislabeling in milk and dairy products

“…During the last decade, the PCR-RFLP technique has been widely used for species identification in meat and dairy products (Branciari et al, 2000;Pfeiffer et al, 2004;El Rady and Sayed, 2006;Fajardo et al, 2006); however, if dairy products are prepared by mixing milk from 2 or more species, the interpretation of PCR-RFLP is almost impossible because of the overlap of restriction patterns (Bottero et al, 2003;Dalmasso et al, 2012). Recently, sensory analysis combined with PCR (Golinelli et al, 2014), allelic discrimination (Dalmasso et al, 2011(Dalmasso et al, , 2012, high-resolution melting analysis (Sakaridis et al, 2013), and analysis of short species-specific mitochondrial DNA targets (Cottenet et al, 2011;Gonçalves et al, 2012) have been proposed as new and interesting methods that may be used in species identification of dairy products, but none of them used a cut-off of 1% that unambiguously differentiates between unintentional and fraudulent contamination with cow milk, as reported in the Commission Regulation (EC) No. 273 of 5 March 2008(European Commission, 2008.…”

“…Besides mass spectrometry techniques (Linder et al, 2010;Calvano et al, 2013), different authors have proposed molecular assays to detect bovine milk in dairy products (López-Calleja et al, 2007b,c;Mafra et al, 2007;Zhang et al, 2007;Cottenet et al, 2011;Dalmasso et al, 2012;Rentsch et al, 2013;Sakaridis et al, 2013), but only Gonçalves et al (2012) and Agrimonti et al (2015) have developed a method for the simultaneous identification of milk from cow, sheep, goat, and water buffalo as described in the present work. The analysis of short species-specific mitochondrial DNA targets proposed by Gonçalves et al (2012) represented a reliable alternative method to real-time PCR even though it was more expensive and time consuming.…”

Duplex PCR approach for the detection and quantification of donkey, horse and mule in raw and heat‐processed meat products

“…The fraudulent substitution or undeclared adulteration of meat species in comminuted and highly processed meat products could be dangerous for people who have allergies to certain types of meat proteins. For example, donkey milk has been selected as a substitute for children below the age of three allergic to cow's milk protein because donkey's milk is much more similar to human milk in composition and palatability (Dalmasso et al, 2012). The addition of other less expensive milk (cow's, goat's or sheep's) to donkey's milk may nullify these products' therapeutic function.…”

Investigation of milk origin in sheep and goat cheese with Real-Time PCR

“…Recently, food safety, quality and composition have become an important issue for consumers, and for the consumers of today, the authenticity of food has an important place. Therefore, especially in animal products, identification of animal species has gained increasing importance (Dalmasso et al, 2012). Consumers are expected to believe the vendors and what is written on the labels because the authenticity of some products is not directly observable (Mayer et al, 2012).…”