Accurate and simultaneous detection of pork and horse meat adulteration by double tailed recombinase polymerase amplification integrated with SERS based two-color lateral flow nucleic acid hybridization strip.

Meat adulteration is a growing concern for consumers and researchers, necessitating the development of a simple and robust method for identifying animal sources in meat products. In this study, a double-tailed recombinase polymerase amplification (RPA) integrated with surface-enhanced Raman scattering (SERS) based two-color lateral flow nucleic acid hybridization strip (LFNAHS) was established for simultaneous detection of pork and horse meat. Magenta and cyan gold nanoparticles functionalized with specific probes and Raman dyestuff were used to color the LFNAHS based on RPA amplification products. The assay successfully detected colored test lines for horse and pork adulteration, with quantitative analysis achieved through Raman intensity measurements. The method showed a high specificity with no cross-reactivity and a detection limit of 0.01 % (wt%). Authenticity testing of 40 donkey products yielded results consistent with PCR-agarose gel electrophoresis. This assay can be extended to identify other meat sources by replacing the specific primer sets. This versatile method can be adapted for identifying other meat sources, making it a valuable tool for accurate, sensitive, and quantitative meat authentication in resource-limited settings. [Display omitted] • A new double tailed mRPA with a RPA-SERS-LFNAHS was developed for donkey authentication. • Magenta spherical and cyan urchin AuNPs were synthesized as multicolor labels in the RPA-SERS-LFNAHS. • The specific RPA primers are uniquely designed with tail sequence and a spacer C9. • The detectability of this mRPA-mNAH-LFS assay for horse meat and pork were 0.01 %. • This assay was successfully applied to the authentication of real donkey products. [ABSTRACT FROM AUTHOR]