A 3D-printed smartphone-based platform for in-situ and rapid monitoring of aquaculture pathogens using polydimethylsiloxane (PDMS) microchip with multiplex loop-mediated isothermal amplification (M-LAMP)

Aquaculture pathogens pose serious risks to aquatic livestock and global food safety. Key threats in shrimp farming include white spot syndrome virus (WSSV), Vibrio parahaemolyticus (causing acute hepatopancreatic necrosis disease, AHPND), and Enterocytozoon hepatopenaei (EHP). Rapid, on-site detection is critical for early detection and outbreak prevention. In this study, we developed a portable, smartphone-based diagnostic platform utilizing multiplex loop-mediated isothermal amplification (LAMP) for simultaneous detection of multiple pathogens in a single reaction. A PDMS microchip with 30 reaction wells (5 × 6 array) and a temperature control well was fabricated for efficient multiplexing. Immobilized LAMP reagents with freeze-drying lyophilization were preloaded into wells to streamline preparation and enhance stability. The system successfully identified both waterborne indicator bacteria (E. coli, E. faecalis, Salmonella) and major shrimp pathogens (WSSV, AHPND, EHP) in samples from Penaeus vannamei, Penaeus monodon, and aquaculture water. The microchip maintained stable isothermal conditions (65.1 ± 0.6 °C), enabling visual detection via color change at low DNA concentrations (as low as 4 copies/μL). All WSSV and EHP infections in shrimp tissues and water samples were correctly identified using LAMP reaction within 35 min (excluding the DNA extraction process), demonstrating 100% positive detection rates. The smartphone interface allowed real-time imaging and result interpretation, offering a rapid, user-friendly tool for in situ pathogen monitoring. This platform represents a practical, low-cost solution for field diagnostics and improved disease management in aquaculture.