Paper microfluidic LAMP-CRISPR/enAsCas12a platform for multiplex detection of waterborne pathogens in drinking water

Pathogenic contamination poses a threat to drinking water safety and public health. To address contamination by Escherichia coli (E. coli), Salmonella spp., and Shigella spp., this study developed paper microfluidics for on-site multi-target detection based on an integrated LAMP-CRISPR/enAsCas12a assay. The device integrates nucleic acid enrichment, purification, elution, and multi-channel reaction processes into a paper-based device. Structural parameters, including hydrophilic pore size, glass fiber adsorption units, reaction chamber dimensions, and plate thickness, were optimized to improve nucleic acid recovery and fluid distribution. CRISPR/enAsCas12a-mediated sequence verification was incorporated to improve endpoint interpretation following LAMP amplification under complex water-matrix conditions. The limits of detection were 1.11 × 10², 1.87 × 10⁴, and 1.95 × 10² copies/μL for E. coli, Salmonella spp., and Shigella spp., respectively, and the maximum DNA recovery efficiency was approximately 70%. Validation using samples from 15 drinking-water source sites in Tongren City, China, generated positive E. coli responses at all sites and Salmonella responses at selected sites, whereas Shigella was not detected under the applied conditions. Comparison with qPCR showed broadly consistent concentration patterns for E. coli, whereas differences were observed for some low-abundance Salmonella samples, which may be associated with the relatively higher detection limit of the Salmonella channel. Overall, the developed platform provides an integrated and portable-compatible approach for decentralized multiplex screening of waterborne pathogens and establishes a basis for further field-oriented development.