Rapid quantification of fecal indicator bacteria in water using the most probable number - loop-mediated isothermal amplification (MPN-LAMP) approach on a polymethyl methacrylate (PMMA) microchip

Abstract

Fecal contamination of water and its associated pathogens are a major public health concern in both developing and industrialized areas. Fecal indicator bacteria (FIB) are commonly used to assess microbial water quality, but they require a relatively long period of incubation time. Currently, molecular techniques have been applied to rapidly detect FIB. However, these molecular techniques require expensive and sophisticated equipment. In this study, we developed a rapid on-chip gene quantification method based on loop-mediated isothermal amplification (LAMP) PCR. The LAMP assays can measure the target genes of the fecal indicator bacteria (FIB), including E. coli and Enterococcus spp, using the most probable number (MPN) approach. The colorimetric LAMP assay allows for naked-eye observation of the PCR reaction as few as 4 gene copies / well. When the reaction ends, MPN measurement of positive outcomes on the white-based PMMA (polymethacrylic acid) microchips provides the concentrations of the target genes of FIB with a confidence interval. We validated the feasibility of the MPN-LAMP approach by obtaining a strong correlation between the results of the MPN estimations and the qPCR analysis. Moreover, the MPN-LAMP approach was used to quantify the FIB in different environmental water collected from the freshwater reservoirs, beach, agriculture farm, and sewage. Our research demonstrates that the MPN- LAMP method enables us to easily and quickly quantifying FIB genes isolated from the environment without expensive qPCR instruments.

Keywords: Fecal indicator bacteria; Loop-mediated isothermal amplification; Most probable number; PMMA microchip.