A real-time LAMP-based dual-sample microfluidic chip for rapid and simultaneous detection of multiple waterborne pathogenic bacteria from coastal waters

Abstract

Waterborne pathogens are becoming a serious worldwide health hazard; thus, the regular monitoring of epidemic pathogens is urgently required for public safety. In the present study, we developed a microfluidic chip integrated loop-mediated isothermal amplification technique (on-chip LAMP) to simultaneously detect 10 waterborne pathogenic bacteria, Campylobacter jejuni, Listeria monocytogenes, Salmonella enterica, Shigella flexneri, Staphylococcus aureus, Vibrio alginolyticus, V. cholerae, V. parahemolyticus, V. vulnificus, and Yersinia enterocolitica. This method was capable of simultaneously completing 22 genetic analyses of two specimens and achieved limits of detection ranging from 7.92 × 10⁻³ to 9.54 × 10⁻¹ pg of genomic DNA of pure bacteria per reaction. The processes from sample loading to microfluidic operation were in a highly automated format, and the LAMP reaction ran to completion within 35 minutes, with a minimal volume of 22 μl per each half of a single chip. The coefficient of variation for the time-to-positive value was less than 0.1, indicating an excellent reproducibility of the dual-sample on-chip LAMP assay. The clinical sensitivity and specificity in analyses of coastal water samples were 93.1% and 98.0%, respectively, in comparison with traditional microbiological methods. Our established dual-sample on-chip LAMP assay provides an effective multiple-pathogen analysis of waterborne bacterial pathogens. This indicates that the method is applicable for on-site detection and routine monitoring of waterborne bacteria in aquatic environments.