Development of a Universal Forward Primer and Species-Specific Reverse Primers for Porcine, Canine, and Murine Gene Detection using SYBR Green Multiplex Quantitative Polymerase Chain Reaction

INTRODUCTION: The growing awareness regarding animal-derived content in pharmaceuticals has led to an increased demand for labeling of animal-derived ingredients. Multiplex qPCR can amplify more than one target gene by combining two or more primer sets in one reaction. Thus, this study focused on developing a universal forward primer and specific reverse primers targeting 16S rRNA for the simultaneous detection of Murine, Porcine, Canine, and Murine. METHODS: These primers were evaluated in silico, followed by in vitro optimization using intercalating dye-based qPCR for detecting the presence of these genes in total DNA extracted from food-based meat and pharmaceutical products. RESULT: These primers successfully produced amplicons in multiplex qPCR with distinct melting temperatures. Additionally, the developed primers in multiplex qPCR were capable of identifying Murine, Canine, and Porcine DNA at concentrations of 10-100 pg with an efficiency of 90- 110%. Repeatability testing revealed a variance of less than 10% for both intra- and inter-assay. Furthermore, the new primer combination successfully detected DNA remnants in positive porcine pharmaceuticals and cosmetics. DISCUSSION: The developed primers were able to differentiate animal species concentrations found in pharmaceuticals and cosmetics with good repeatability. However, porcine peaks in sample analysis were still low due to the low yield of DNA extraction using a food-grade DNA extraction kit Conclusion: These results suggest that the new primer combination, consisting of the universal forward primer and species-specific reverse primers, has the potential to serve as an alternative assay for differentiating canine, porcine, and murine DNA using multiplex intercalating dyebased qPCR.