We have been developing an innovative platform to detect Escherichia coli outbreaks for monitoring water quality. E. coli&nbsp;are a large and diverse group of bacteria. Most strains are harmless, but others can cause abdominal cramps, vomiting, and diarrhea. Infection can occur even by small amounts of contaminated water. In addition, the presence of E. coli is a water quality indicator recommended by the United States Environmental Protection Agency (US EPA) since E. coli are present in fecal material and fecal pollution is the main source for disease‐causing agents in water. Moreover, the presence of E. coli over recommended levels can indicate the presence of other pathogenic organisms.For detecting E. coli&nbsp;in water in the field, we have developed a device that carries out both sample preparation and detection without requiring laboratory tools. Our device consists of four parts, including one buffer unit, one mixing unit, one waste container, and one detection unit. The buffer unit consists of a large opening and 4 reservoirs for a lysis buffer, a binding buffer, and two washing buffers. The detection unit is an assembly of two components: a chromatography paper pad that is laminated between two thermoplastic films; and a polycarbonate well layer. The device is designed to pass a water sample through a funnel-shaped reservoir in the mixing unit, and then through the paper filter to concentrate DNA on paper in the detection unit, into the waste container at the bottom. The sequential delivery of reagents needed for sample preparation is achieved by ball-based valves that are actuated by rotating the buffer unit at the top. The opening at the bottom of each reservoir is blocked by a stainless-steel ball to prevent the reagents from flowing down until they are desired. When the buffer unit is being rotated, the ball at the bottom of one reservoir will be aligned with the pin inside the mixing unit, which lifts up the ball to allow the solution in that reservoir to flow into the mixing unit.&nbsp; After passing the paper in the detection unit, all solutions are collected in the waste container.The collected DNA is then amplified by using a smart coffee mug (Ember Travel Mug) that provides constant temperature for loop-mediated isothermal amplification (LAMP), followed by colorimetric detection. The colorimetric detection is carried out by using SYBR Green dye after amplification. &nbsp;After theoretical analysis and experimental optimization, we have incorporated a suction mechanism into the device to speed up the sample preparation, which may also be facilitated by enlarging the size of the filter paper.We have demonstrated the functions of the device by detecting several E. coli strains including DH5-α. We have determined the limit of detection of our LAMP assay as 3 genome equivalents using purified E. coli DH5-α DNA. In addition, we have demonstrated detection of E. coli in recreational water samples collected during Hurricane Dorian.In summary, we have developed an on-site testing platform capable of detecting E. coli in water samples in just 90 minutes, which is much faster than traditional cell-culture-based approaches that usually take 1-2 days. Our platform offers high sensitivity, speed, easy-to-use, and portability for the field use.

Miniaturized Platform for On-Site Detection of E. Coli in Water Samples

Paper Proceedings: https://asmedigitalcollection.asme.org/FEDSM/FEDSM2021/volume/85284

ASME 2021 Fluids Engineering Division Summer Meeting (FEDSM2021)

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