A point-of-care testing platform for pathogen nucleic acid analysis utilizing paper-based extraction and portable qPCR.

Rapid and reliable diagnostic methods are crucial for controlling animal disease outbreaks and reducing mortality, especially when effective drugs or vaccines are unavailable. While real-time quantitative polymerase chain reaction (qPCR) remains the gold standard for nucleic acid detection due to its superior sensitivity and specificity, conventional workflows are inherently cumbersome. Traditional extraction and amplification processes typically require 1.5 to 3 h, with further delays incurred by transporting samples to centralized laboratories. To overcome these limitations, this study introduces a point-of-care testing platform for on-site pathogen diagnostics. This system synergistically combines a centrifuge-free, glass fiber (GF) paper-based nucleic acid extraction card, room-temperature-stable lyophilized qPCR reagents, and a portable qPCR instrument. By eliminating the dependency on bulky benchtop equipment and cold-chain logistics, this platform streamlines lysis, extraction, and amplification, delivering sample-to-answer results within 45 min. The optimized system demonstrated excellent analytical sensitivity, achieving limits of detection (LoDs) of 10 copies/μL for the N gene of porcine epidemic diarrhea virus (PEDV) and 50 copies/μL for the PirA gene of Vibrio parahaemolyticus AHPND (VPAHPND). Furthermore, clinical validation using complex field samples yielded a 100% diagnostic concordance rate with standard laboratory equipment. This study highlights the potential of the proposed system for early diagnosis and monitoring of animal diseases, providing a promising solution for rapid on-site screening in resource-constrained settings.