Surface-engineered paper-based microfluidic platform for fully automated RPA detection of swine viral diseases

The rapid and accurate detection of swine viral diseases such as African swine fever (ASF) and pseudorabies (PR) is critical for preventing large-scale outbreaks and minimizing economic losses in the livestock industry. Among nucleic acid amplification methods, recombinase polymerase amplification (RPA) is particularly well-suited for point-of-care detection of swine viruses, as it is isothermal and delivers results within 20 min. Microfluidic paper-based analytical devices (µPADs) offer a promising platform for developing point-of-care testing RPA devices, offering low cost, ease of use, and minimal sample consumption. However, existing paper-based RPA systems still face practical challenges, including insufficient automation and slow transport of reagents caused by the incorporation of viscous additives (e.g., PEG) for the RPA reaction within paper matrices. To address these limitations, we implemented a surface-modification approach to accelerate fluid transport and developed a fully automated μPAD for RPA-based ASFV and PRV screening. This approach enhanced capillary-driven flow, cutting reagent transfer time from 82 s to 27 s (67 % reduction). In addition, enhanced flow performance did not appear immediately after fabrication and instead transitioned from reduced efficiency on Day 1 to a stable operational window from Day 3 to Day 11, ensuring reliable operation under its intended use conditions. This “sample-in, answer-out” system eliminates the need for external equipment, achieving detection limits of 10 copies/μL for both ASFV and PRV within 11.5 and 14.5 min, respectively, demonstrating high sensitivity and rapid results in a self-contained, portable, and low-cost format, which offers a step forward for practical, on-site viral diagnosis in resource-limited environments.