Development of fast multiplex real-time PCR assays based on EvaGreen fluorescence dye for identification of beef and soybean origins in processed sausages

EvaGreen multiplex real-time polymerase chain reaction (EMRT-PCR) was designed for an assay that can unite the advantages of real-time PCR and multiplex PCR to recognize animal genes more quickly in sausages. EMRT-PCR based on melting temperatures discrimination by using EvaGreen fluorescence dye was developed for the analysis of beef and soybean in sausages. The method combines the use of bovine (Bos taurus) and soybean (Glycine max) specific primers that amplify small fragments (bovine; 271 bp and soybean; 100 bp) of the mitochondrial bovine ATPase8 subunit and lectin genes, respectively. Appropriate mixtures of beef and soybean sausage DNA were used to develop the assay. Gene products of beef (bovine) and soybean were represented in two melting peaks generated simultaneously at 82 °C and 86 °C, respectively. Analysis of the reference sausages showed that the detection limit of the assay was 0.003% for beef and 0.001% for soybean species. Multiplex results obtained with one of the multiplex polymerase mix linked extremely well with the uniplex reference. The aim of the study was to design a fast, sensitive, specific and accurate multiplex real-time PCR assay by using EvaGreen fluorescence dye that is cost-effective than double labeled probes to detect the beef and soybean species in sausages simultaneously.