Versatile dual-channel loop-mediated isothermal amplification assay featuring smartphone imaging enables determination of fecal indicator bacteria in environmental waters

Abstract

Rapid diagnostic assays are often a critical tool for monitoring water quality in developing and developed countries. Conventional testing requires 24–48 h for incubation, resulting in delayed remediation and increasing the likelihood of negative outcomes. In this study, we report a workflow for detection of E. coli, a common indicator of fecal contamination. Following large volume filtration, E. coli is then solubilized enabling the facile isolation and recovery of genetic material by a thin film microextraction (TFME) device featuring a polymeric ionic liquid (PIL) sorbent. Rapid recovery of pure nucleic acids is achieved using a PIL sorbent with high affinity for DNA to significantly increase mass transfer and facilitate adsorption and desorption of DNA. Downstream detection is performed by a versatile, dual channel loop mediated isothermal amplification (LAMP) assay featuring a colorimetric dye and a sequence-specific molecular beacon. A portable LAMP companion box enables consistent isothermal heating and endpoint smartphone imaging while being powered by a single 12-V battery. Programmable LEDs are switched from white or blue light to facilitate the independent imaging of the colorimetric dye or fluorometric probe following amplification. The methodology positively identified E. coli in environmental samples spiked to concentrations of 6600 colony forming units (CFU) per milliliter and 660 CFU/mL with 100% and 22% positivity, respectively.