Ultrasensitive Detection of Porcine Epidemic Diarrhea Virus Infections Using Multivalent DNA Nanostructure‐Enabled Lateral Flow Assay

Early, rapid on-site detection of viral pathogens in livestock is critical to prevent disease spread, minimize economic losses, and safeguard food security. However, most current diagnostics are limited to centralized laboratories or require specialized equipment such as electrochemical readers, with a few hours to multi-day turnaround times that hinder field deployment. Here, we introduce a DNA nanostructure-enabled lateral flow assay (LFA) for rapid, ultrasensitive detection of porcine epidemic diarrhea virus (PEDV) directly on farms. This assay employs net-shaped DNA nanostructures presenting PEDV-specific aptamers in a multivalent configuration, yielding ∼1000-fold greater binding avidity than free aptamers. The platform demonstrates the limit of detection (LOD) of ∼1.33 ng/mL for recombinant PEDV nucleocapsid protein and ∼278 viral copies per test in pig oral fluid samples, with results obtained within 10 min without requiring nucleic acid extraction or amplification. Validation with animal clinical samples (n = 102) from swine farms demonstrates an accuracy of 97.06%, sensitivity of 96.92%, and specificity of 97.30% (Ct ≤ 37.42), without cross-reactivity against other swine viruses. This work presents a robust and field-deployable diagnostic platform that combines the programmability of DNA nanotechnology with the operational simplicity of LFA. Beyond PEDV, the modular nature of the DNA-Net platform is readily adaptable for detecting other veterinary and zoonotic pathogens, advancing disease surveillance, and One Health diagnostics.