A polymer microfluidic chip for quantitative detection of multiple water- and foodborne pathogens using real-time fluorogenic loop-mediated isothermal amplification

Abstract

Inexpensive, portable, and easy-to-use devices for rapid detection of microbial pathogens are needed to ensure safety of water and food. In this study, a disposable polymer microfluidic chip for quantitative detection of multiple pathogens using isothermal nucleic acid amplification was developed. The chip contains an array of 15 interconnected reaction wells with dehydrated primers for loop-mediated isothermal amplification (LAMP), and requires only a single pipetting step for dispensing of sample. To improve robustness of loading and amplification, hydrophobic air vents and microvalves were monolithically integrated in the multi-layered structure of the chip using an inexpensive knife plotter. For quantification, LAMP was performed with a highly fluorescent DNA binding dye (SYTO-82) and the reactions monitored in real-time using a low-cost fluorescence imaging system previously developed by our group (Ahmad et al., Biomed. Microdevices 13(5), 929-937). Starting from genomic DNA mixtures, the chip was successfully evaluated for rapid analysis of multiple virulence and marker genes of Salmonella, Campylobacter jejuni, Shigella, and Vibrio cholerae, enabling detection and quantification of 10-100 genomes per μl in less than 20 min. It is anticipated that the microfluidic chip, along with the real-time imaging system, may be a key enabling technology for developing inexpensive and portable systems for on-site screening of multiple pathogens relevant to food and water safety.