The cleanliness of natural waterways poses a serious health concern for adjacent ecosystems and provides a breeding ground for human enteric pathogens. Commonly, waterways are surveyed for high fecal contamination produced by livestock or population centers by detecting fecal indicator bacteria (FIB) through microbial cultivation. However, this method requires equipped laboratories and provides results after several days. Recently, molecular detection methodologies based on nucleic acid amplification assays (NAAAs), particularly quantitative polymerase chain reaction (qPCR), have been introduced for the detection of selected FIB such as E. coli. These assays can be significantly hindered by inhibitor molecules, necessitating the purification of nucleic acids for highly sensitive and reliable analysis. Using commercial DNA extraction kits, qPCR allows for rapid detection of FIB but requires expensive instrumentation. Alternative NAAAs such as loop-mediated isothermal amplification (LAMP) require only a constant temperature of 60-65 °C and have been used for the rapid detection of SARS-CoV-2 in low resource settings throughout the COVID-19 pandemic. A workflow featuring isolation and purification of DNA prior to a LAMP assay for the in-field detection of FIB in waterways can be used to provide results within an hour of sampling using smartphone detection. In this presentation, a LAMP assay featuring a dual colorimetric and fluorescent detection method was developed for the determination of FIB E. coil from environmental water. For in-field detection, a user-friendly smartphone-based device featuring a colorimetric channel for detection of positive reactions and a fluorescence channel that enables specific detection of E. coli DNA using a molecular beacon probe was developed. The device consists of a light source and an emission filter contained within a 3D printed housing to reduce outside noise. To eliminate matrix effects and obtain higher sensitivity and reliability through analyte enrichment, the method is combined with solid-phase microextraction.

In-field smartphone determination of Escherichia coli contamination in environmental water using loop-mediated isothermal amplification

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ACS Fall 2022

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