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.