A versatile real-time PCR method to quantify bovine contamination in buffalo products

Fraudulent species substitution in food products is a reality in many markets around the world. The consequences are serious and impact governments, consumers and producers. Molecular methods, including PCR-based techniques, are available to prevent species substitutions and to authenticate food content. Here, we describe a method for calculating the bovine and buffalo content in milk- and meat-derived food products. The method applies real-time PCR using primers designed to specifically amplify bovine or buffalo DNA. The amplification efficiencies of both primer sets were assessed using the TaqMan and SYBR Green systems. Both sets of primers showed satisfactory results. A quantification procedure is proposed and involves amplifying a sample with both primer sets and then normalizing the total DNA using the total non-normalized bovine and buffalo DNA. To correct for the potential deviations between the real and measured DNA quantity caused by biological differences between species, we propose the use of calibration curves generated from each analyzed matrix. These curves include a set of controlled admixtures of bovine and buffalo material. Using this method for dairy samples containing known bovine and buffalo content, the use of the calibration curve always approximated the measured to the expected quantities. This technique was then tested on commercial samples. Our method was efficacious and reliable and can be applied to the routine analysis of these products. Although we only tested the methodology on dairy products, it can also be applied to other food matrices, such as meat-derived products. A patent document for the technique has been submitted to the Brazilian Patent Office (INPI – Instituto Nacional da Propriedade Industrial –www.inpi.gov.br) under the number 014110002844.