Magnetic Nanoparticle-Assisted Multiplex RPA for Rapid Meat Authentication via Fluorescence Detection

Meat adulteration with undeclared species remains a pervasive problem. We report a rapid, sensitive, and field-deployable assay-termed magnetic nanoparticle-assisted multiplex recombinase polymerase amplification (mRPA) with fluorescence detection for the simultaneous detection of horse and chicken components in meat. It integrates mRPA, streptavidin-coated core–shell Fe3O4@SiO2 nanoparticles, and dual-labeled primers targeting ATP6–8 (horse) and CYTB (chicken) genes. Forward primers were labeled with 6-FAM or Cy5, and reverse primers were biotinylated. The assay leverages biotin–streptavidin interactions and magnetic separation (MS) to isolate fluorescent amplicons, effectively reducing background noise and enhancing specificity. Under the optimized conditions, the assay can detect 0.1% (w/w) adulteration without cross-reactivity. Combined with a 96-well DNA extraction module, the workflow could be finished in 31.5 min for 96 samples. In 102 commercial samples, mRPA-MS-FD fully agreed with national-standard RT-qPCR, identifying 14 positives. This platform unites isothermal amplification and MS to deliver sensitive, multiplexed, high-throughput meat authentication for routine surveillance and point-of-need screening.