Empowering Safe Water Access: A Field-Deployable Tool for Rapid Fecal Contamination Detection

Introduction: : The unmet need for technologies that detect fecal matter contamination in water sources is significant, driven by the global challenge of waterborne diseases and the increasing need for safe drinking water. Water contaminated with fecal matter can introduce harmful pathogens such as E. coli, cholera, and typhoid, leading to illness or even death, particularly in developing regions where access to clean water and sanitation infrastructure is limited. Traditional methods for detecting fecal contamination in water, such as culturing techniques (e.g., IDEXX Quanty trays that test coliform bacteria), are often time-consuming, require trained personnel, and may not provide real-time results. This delay can exacerbate health risks, especially in emergencies or in regions lacking infrastructure.
Our technology offers rapid, inexpensive, and easy-to-use nucleic acid extraction and identification to detect pathogenic fecal organisms in environmental samples such as surface/ground waters. This portable system, which only includes a 50 mL tube filled with reagents, an attachable immiscible filtration assisted by surface tension (IFAST) cleanup chip, and a hotplate, allows on-site and real-time testing, with an estimated sample-to-answer time of ~40 minutes (Figure 1A-B). This technology can be a powerful tool to detect low concentrations of harmful fecal matter contamination (e.g., E. coli) in drinking water sources.

Materials and

Methods: : To replicate contaminated stream water, tap water was spiked with composite wastewater collected from wastewater treatment plants (WWTPs). Also, grab samples were collected from three creek sites in Eastern Kentucky recently affected by flooding and sewage damage. Our standard nucleic acid (NA) standard extraction method started with the addition of 375uL lysis buffer (4M GTC, 0.01M MES, and 1:1 volume of deionized water and absolute ethanol), and 20uL of SeraSil-Mag™ beads to 250uL of the water sample. The samples were then mixed and incubated for 20mins at 50C and 20mins at room temperature. The extraction and washing steps were performed on the Extractman™ (Gilson) platform. Then the NA was eluted in nuclease-free water at 70C for 5mins. The identification was performed using RT-qPCR (Roche) and colorimetric loop-mediated isothermal amplification (LAMP) (NEB). For IFAST cleanup chip fabrication, five sequential wells were laser-cut into a 1.7mm PMMA sheet. The first well was used to firmly attach the chip to a 50mL tube that contains sample mixture (16mL sample + 24mL lysis buffer + 120uL beads), and the other wells contain silicon oil, wash buffer, silicon oil, and nuclease-free water, respectively (Figure 1B). Channels (500um deep) linked the wells, and the chip was sealed with PCR adhesive tape. After connecting the sample mixture tube, the beads were moved through wells via a magnet for purification. Then, the chip was heated at 70C for 5mins to elute NA, which was then collected for identification purposes as mentioned above.

Results, Conclusions, and Discussions:: One of the main challenges of detecting E. coli contamination in surface water is the low concentration of the target in water samples. We increased the final concentration of the extracted nucleic acid by starting with a high sample volume (16 mL) and minimizing the elution volume to 80uL. Integrated IFAST sample cleanup method eliminated the need for complex and expensive equipment, minimized the liquid handling steps, and decreased the total concentration and extraction procedures time to less than 7mins per sample (including the 5mins elution time). The final concentration of the extracted E. coli nucleic acid was increased 20-43 fold (Figure 1C), and the extraction efficiency was 25-55%, compared to our standard extraction method. To identify the E. coli nucleic acid, we developed an isothermal colorimetric LAMP specific to the Uid-A gene, which required minimal equipment such as a hotplate. We achieved a limit of detection (LOD) of 250 DNA copies per reaction in 25uL colorimetric LAMP reaction protocol in less than 30 minutes (Figure 1D). This results in a sample-to-answer time of < 35mins with a sensitivity of < 10 DNA copies per mL of water samples. Also, we validated the performance of our technology against the Quanty tray (IDEXX) system on creek samples, known to be contaminated by sewage due to damage caused by recent floods to the local septic tanks.
The gold standard method (Quanty tray) to detect E. coli in drinking water sources is limited to centralized labs, which increases the cost (tens to hundreds of dollars) and the sample-to-answer time (on the order of days). Our portable nucleic acid-based detection system addresses a critical gap in current water quality monitoring technologies by enabling rapid, low-cost, and on-site detection of fecal contamination. By integrating high-volume sample processing, efficient IFAST-based clean-up, and sensitive isothermal LAMP, the platform significantly reduces both time and resource requirements compared to traditional methods. This approach offers a practical and scalable solution for real-time water safety monitoring, particularly in resource-limited and emergency settings.

Acknowledgements and/or References (Optional):: The work was supported by the National Institutes of Health (NIH) grants 1U01DA053903-01 and P30 ES026529, the Centers for Disease Control and Prevention (CDC) contract BAA 75D301-20-R-68024, and National Science Foundation (NSF) grant 2154934 and 2412446.