Abstract
The increasing prevalence of aquatic sports presents significant public health concerns due to the risk of infections from waterborne pathogens. Conventional detection methods are often labor intensive, rely on sophisticated laboratory instrumentation, and are inadequate for meeting the urgent demand for onsite, point-of-care testing (POCT). This study reports the development and validation of an integrated POCT platform. The system incorporates three core modules: (1) an instrument-free, syringe-actuated device for simple nucleic acid extraction; (2) a PMMA microfluidic chip enabling uniform sample distribution to multiple reaction chambers via manual centrifugation; and (3) a visual, pH indicator-based loop-mediated isothermal amplification (LAMP) assay. The primers and reaction chemistries were systematically optimized for pathogens, including Staphylococcus aureus and Shigella flexneri. The developed platform enables a sample-to-answer workflow in approximately 60 min, with an analytical limit of detection of 10-100 copies per reaction. Notably, the microfluidic chip platform showed enhanced performance compared to conventional tube-based assays in both sensitivity and stability, particularly yielding more robust results for low-titer samples. Furthermore, the simplified extraction method achieved a recovery efficiency for gram-negative bacteria comparable to that of commercial kits, while noting differences in efficiency for gram-positive strains. The integrated platform exhibited high specificity and robustness against interferents in simulated contaminated water. This work demonstrates a user-friendly, cost-effective, and fully integrated platform for the visual detection of multiple pathogens, requiring only a portable heating module for its entire operation. The platform not only offers a viable technical solution for onsite safety monitoring in aquatic sports but also validates the concept of "backend compensation": a well-designed detection module can help offset the performance loss from simplified frontend sample preparation. This philosophy provides valuable insights for developing reliable POCT systems destined for real-world, resource-limited settings.