Enhancing Food Transparency: PCR-HRM-Based Multiplex Assay for Simultaneous Detection of Four Animal Species in Food

To address the growing global issue of food fraud through animal species substitution, a quadruplex polymerase chain reaction combined with a high-resolution melting assay was developed in a single tube to rapidly identify the cytb DNA of four animal species. Custom primers were meticulously designed and experimentally validated; the Tm values for porcine, bovine, donkey, and tilapia amplicons were 72.28, 76.25, 79.32, and 84.14 °C, respectively. The results demonstrated that the method possessed high specificity (8:8), with a template DNA detection limit as low as 0.05 ng/μL, and was capable of reliably identifying 1% porcine adulteration in diverse food matrices. Moreover, this method was employed to detect animal-derived constituents within diverse processed food samples. The PCR-HRM assay was a rapid, cost-effective, and sensitive tool for food authenticity verification.