Lateral flow biosensor utilizing self-assembled DNA-multicolor Au/Ag nanoassemblies and dual-tailed RPA for on-site triplex detection of chicken duck and pork adulteration in beef

Meat adulteration has become a significant concern due to its potential to diminish the nutritional value and trigger allergic reactions. To address the issue of fraudulent adulteration in beef ( Bos taurus ) products, a visual strategy is proposed for multiplex detection of chicken ( Gallus gallus ), duck ( Anas platyrhynchos ), and pork ( Sus scrofa ) components in beef. First, the DNA could be easily extracted from the meat samples using the microneedle within 1 min and used as recombinase polymerase amplification (RPA) template. Next, three primer pairs specific to chicken, duck, and pork DNA are designed with unique oligonucleotide tags and a spacerC9 to halt polymerase activity, enabling the RPA amplicons to possess a single-stranded DNA tail. These tail sequences precisely hybridize with nanoparticle-labeled detection probes and pre-immobilized capture probes on test lines, generating a colorimetric signal on the lateral flow strip (LFS). The enhanced LFS utilizes three distinct Au/Ag nanoparticles, including Au@Ag (yellow), gold nanoflowers (cyan), and gold nanospheres (magenta), each targeting chicken, duck, and pork DNA, respectively. The test output can be easily interpreted through a “magenta yellow cyan” color code. The sensitivity of the multicolor LFS is further improved by the incorporation of DNA-Au/Ag nanoaggregates. Four functional oligonucleotides with complementary sequences are combined to form DNA-Au/Ag nano aggregates, allowing for increased coupling of Au/Ag and enhancing detection sensitivity. Cross-reactivity tests with ten non-target species demonstrate the excellent specificity of the assay. The method can detect chicken, duck, or pork at levels as low as 0.1 g/kg in simulated adulterated beef samples. The entire process, including rapid DNA extraction (1 min), RPA reaction (15 min), and LFS detection (5 min), is completed within 21 min. This platform is user-friendly, eliminating extensive training and specialized equipment, making it ideal for rapid on-site detection of multiplex adulteration by personnel in various fields. • A 21-min detection workflow was developed that integrates microneedle-based DNA extraction, RPA, and multicolor NAHLFS. • Multicolor coding (magenta/yellow/cyan) enables simultaneous identification of chicken, duck, and pork. • The method can detect chicken, duck, or pork at levels as low as 0.1 g/kg in simulated adulterated beef samples. • The RPA-NAHLFS assay showed no cross-reactivity with nine non-target meat species and one vegetable species. • A field-deployable workflow facilitates on-site authentication by non-expert operators.