Development of a duplex recombinase polymerase amplification-lateral flow strip assay with 1 min of DNA extraction for simultaneous identification of pork and chicken ingredients

Meat adulteration is a challenge faced by the global food industry. However, existing protein-based methods or DNA-based polymerase chain reactions are time-consuming and require specialist devices. Therefore, a rapid and on-site assay is urgently needed for identifying meat species. In this study, we developed a duplex recombinase polymerase amplification-lateral flow strip (dRPA-LFS) assay coupled with a rapid DNA extraction method (utilizing the UiO-66 coated stick as the DNA isolation device, and the isolation can be completed in 1minute, including lysis, washing, and desorption in three steps) for the identification of pork and chicken ingredients. Targeted genes, the pork (Porcine) mitochondrial ND2 gene and chicken (Gallus gallus) cytochrome B gene, were designed with primers and probes. The whole dRPA-LFS detection procedure can be finished within 18.5minutes (including 1minute of rapid DNA extraction, 15minutes of dRPA amplification, and 2.5minutes of LFS semi-quantitative detection by the naked eye), and the result can also be quantified by ImageJ software within 15minutes. No positive amplification was observed in beef, lamb, duck, rabbit, goose, ostrich, and horse meat DNA. This assay is sensitive and can detect as low as 0.1% (wt%) of pork and chicken in simulated adulterated meat mixtures. This assay was successfully applied to the authentication identification of 30 commercial beef and lamb products.