Simultaneous detection of cow and buffalo species in milk from China, India, and Pakistan using multiplex real-time PCR

Abstract

Asian countries are major producers of cow and buffalo milk. For quality and authenticity purposes, a multiplex real-time PCR assay was developed to specifically and simultaneously detect DNA from these 2 bovine species. Targeting the cytochrome b gene of mitochondrial DNA, common PCR primers amplified a 105-bp fragment, and 2 fluorescent probes specific to either cow or buffalo were designed for their identification. Specificity was successfully tested on 6 other species, including sheep and goat, and sensitivity reached 1% of cow DNA in buffalo DNA and vice versa. As an evaluation, the method was tested using 119 freeze-dried Asian milk samples from regional industrial milk facilities. Although these samples did not cover the entire Asian zone, the multiplex assay indicated that approximately 20% of the samples (mainly from India) showed high levels of cross-contamination of cow milk by buffalo milk, and vice versa. Fast, sensitive, and straightforward, this method is fit-for-purpose for the authenticity control of Asian milk.

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.…”

“…However, currently, DNA techniques are largely applied for species identification because they have proven to be reliable, specific, sensitive, and fast. In particular, real-time PCR does not require any postamplification step and can be easily automated, allowing the analysis of large numbers of samples (López-Calleja et al, 2007a,b;Cottenet et al, 2011;Dalmasso et al, 2011;Rentsch et al, 2013;Iwobi et al, 2015), and it permits quantitative or semiquantitative analysis.…”

Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique

“…To fulfill the multiplex detection, the obstacle was to develop different primers and probes to simultaneously perform well in one reaction of real-time PCR. No previous research had shown that the triplex real-time PCR with endogenous control was developed for the detection of adulteration in milks and dairy products (López-Calleja et al, 2007a,b;Cottenet et al, 2011;Dalmasso et al, 2011). Specificity assay results in the triplex real-time PCR showed that the primers and probes could cooperate in one reaction for the identification of bovine and equine DNA in milks and dairy products.…”

Proteomics method to quantify the percentage of cow, goat, and sheep milks in raw materials for dairy products

“…Some methods have been developed to evaluate milk authenticity. A DNA-based PCR method is widely used for testing milk authenticity (Abdel-Rahman and Ahmed, 2007;López-Calleja et al, 2007a;Cottenet et al, 2011), but it cannot quantify the percentage of each type of milk because the DNA content in milk is highly variable depending on the health of the cow (Raynal-Ljutovac et al, 2007). Protein-based ELISA is another widely adopted method (López-Calleja et al, 2007b;Costa et al, 2008;Song et al, 2011), and the ELISA is based on specific antibody-antigen reactions.…”