Optimization of multiplex PCR for the identification of animal species using mitochondrial genes in sausages

Detection of species fraud in meat products is very important in order to protect consumers from undesirable adulteration, as well as for the economic, religious and health aspects. The most important reason for verification of the labeling statements is to detect fraudulent substitution of expensive meat components with other cheaper animals or mislabeling. The aim of this study was to develop a multiplex PCR that could be used in the simultaneous identification of multiple meat species. In this study, ten sausages with a minimum beef content of 55 %, from ten different manufacturing companies, and five samples of cow, chicken, goat, camel and donkey raw meats, for the purpose of positive control, were collected from food markets in Tehran, Iran. Total DNA was extracted from each sausage and the raw meats. Primers were selected in different regions of mitochondrial DNA (12S rRNA, cytochrome b and NADH dehydrogenase subunits 2) for identification of meat species. 12S rRNA and NADH dehydrogenase subunits 2 primers generated specific fragments of 183 and 145 bp length, for chicken and donkey, respectively. Three different specific primers were used for amplification of cytochrome b gene in goat, camel and cattle species and amplified species-specific DNA fragments of 157, 200 and 274 bp, respectively. The results proved that half of the specimens were contaminated with chicken meat, and this was greater than the proportion of beef stated on the label, while the other half only had chicken residuals, and no beef content. No contamination was found with goat, donkey or camel meats. These findings showed that molecular methods, such as multiplex PCR, is a potentially reliable, sensitive and accurate assay for the detection of adulterated meat species in mixed meat products.