Abstract

Meat adulteration poses a significant threat to food safety and consumer confidence. Duck is often used as a low-cost alternative, while donkey and sheep/goat meat are frequently fraudulently replaced due to their higher market value. These practices undermine consumer trust and complicate regulatory oversight. In this study, we developed a rapid and reliable method for identifying these species. Through bioinformatics analysis, species-specific nuclear single-copy genes were selected as targets, namely NC_051786.1 for duck, NC_052185.1 for donkey, and NC_030825.1 for sheep/goat. Based on these sequences, primers and probes were designed to establish enzymatic recombinase amplification (ERA) assays, which were coupled with lateral flow strip (LFS) technology for rapid, on-site meat authentication. The ERA-LFS assays operated at 39–41 °C, showed high specificity with no cross-reactivity to non-target species, and achieved a detection limit of 0.016 ng genomic DNA per reaction, with results obtained within 20–30 minutes of reaction time. In spiked genomic DNA samples, the assays successfully detected as low as 0.5% duck and donkey DNA and 0.1% sheep DNA. Furthermore, the assays were validated through testing a range of commercially available processed meat products. These results demonstrate that the developed ERA-LFS assays provide a rapid, sensitive, and specific approach for meat species authentication, offering a practical tool for routine monitoring, on-site inspection, and regulatory enforcement to safeguard food safety and consumer health.