Identification of donkey meat in foods using species-specific PCR combined with lateral flow immunoassay

Food authenticity is a global issue and has raised increasing concerns in the past decades. DNA-based methods are more favourable than the conventional protein-based techniques and have been applied to species identification and meat fraud detection. To effectively identify donkey meat for meat product authentication, a highly specific and robust method that coupled polymerase chain reaction (PCR) with lateral flow immunoassay (LFI) was developed. Donkey-specific PCR primers were designed by targeting at the mitochondrial D-loop gene and the specificity was verified in silico and in vitro against 22 species involved in meat authentication. A limit of detection of 0.0013 ng μL^-1 DNA extract was achieved and as low as 0.001% w/w (raw) and 0.01% w/w (cooked) donkey meat in beef were successfully detected using the developed PCR-LFI. LFI strip-based visualization of PCR products allowed for a 10-fold higher sensitivity than conventional gel electrophoresis and significantly reduced the analysis time for the post-PCR analysis. This PCR-LFI is highly suitable for rapid identification of donkey or incorporating into multiplex screening protocol for other meat authentication in the laboratories of both regulatory agencies and commercial services.