M. Han, G. Kamalakshakurup, A.P. Lee, S. Jiang
University of California, Irvine,
Keywords: digital droplet LAMP, microfluidic chip, Enterococcus
Summary:Rapid and sensitive monitoring of water quality can effectively prevent accidental human exposure to waterborne pathogens. In this project, a rapid quantification method was developed for Enterococcus faecalis, a fecal indicator bacteria (FIB), used as a surrogate for water quality assurance. The loop-mediated isothermal amplification (LAMP) assay was combined with a microfluidic chip to produce thousands of individual reactions that convert the qualitative LAMP assay to quantitative results base on Poisson distribution. This "Lab on a Chip" (LOC) assay is considerably more rapid than the conventional culture-based and the polymerase chain reaction (PCR) based methods (i.e. qPCR and droplet digital PCR). The isothermal amplification removes the need of thermal cycling, which makes it suitable for field development. In this assay, the water sample is mixed with LAMP reagents in the presence of fluorescent dye. The mixture is then dispersed into thousands of droplets in oil on a microfluidic chip, where each droplet acts as an individual LAMP reaction. By counting the number of positive droplets among total droplets in the viewing field, the most probable number of the E. faecalis can be quantified statistically. Several types and compositions of oil were tested for the compatibility with sample/LAMP reagent mixture for droplet generation and stability. The optimized oil can form unified droplets of 70 µm in diameter, which were stable during LAMP reaction at 65℃ for 30mins. The droplets that stained with dsDNA fluorescent dye could be clearly visualized using fluorescence microscope and counted by Image J software. The limit of detection of droplet LAMP assay is 2cfu/ul with pure culture, and 3cfu/ul in seawater.