Reliable multiplex real-time PCR method for detecting adulteration in processed beef products

The screening and identification of meat components has become a critical research area because of the increasing incidence of meat adulteration scandals worldwide. In this study, we developed a multiplex real-time Polymerase Chain Reaction (PCR) method for the detection of beef, pork, chicken, and duck meat. The nuclear genes tumor necrosis factor receptor superfamily member 10 A (TNFRSF10A), prion protein (PRNP), transforming growth factor beta3 (TGF-beta 3), and Beta-actin (ACTB) were selected as target genes. Primers and probes were designed based on the species-specific sequences of these genes to establish a multiplex real-time PCR method. This method demonstrated high specificity, as fluorescence signals were observed only in the target species. To evaluate accuracy, 11 reference samples were tested, and the expected results were observed for all samples. The limit of detection (LOD) for beef, pork, chicken, and duck were 10, 10, 10, and 20 copies, respectively. We applied this method to 34 processed meat products, revealing undeclared meat in a few processed beef products and the absence of beef in two samples labeled as containing beef. Therefore, multiplex real-time PCR is a reliable tool for screening pork, chicken, and duck adulteration in processed beef products.