Rapid assay for multiplexing waterborne pathogens in groundwater

Groundwater serves as a primary drinking water source for half of the global population, yet it remains susceptible to contamination by viral and microbial pathogens originating from agricultural runoff and sewage intrusion. Pathogens such as Escherichia coli, Cryptosporidium, Giardia, and noroviruses are leading causes of waterborne outbreaks, and their detection in groundwater is critical for safeguarding public health. Current monitoring approaches rely heavily on culture-based methods and single-target PCR, all of which are constrained by long turnaround times, limited sensitivity for low-abundance organisms, and an inability to screen multiple pathogens (virus and microbes) simultaneously. Therefore, a multiplexed assay is needed for groundwater surveillance.
This project proposes the development of a rapid assay capable of simultaneously detecting multiple waterborne pathogens within a single test. The assay design integrates three key innovations: (a) pathogen-specific probes targeting bacterial and viral pathogens; (b) multiplexed channels allowing concurrent detection without signal interference; and (c) a streamlined sample preparation workflow compatible with complex groundwater matrices. Together, these features are intended to achieve high analytical sensitivity, specificity, and robustness, while reducing assay time to under two hours.
The research plan consists of two phases. First, we will design the probes to maximize coverage of clinically relevant pathogens, both viral and microbial, while minimizing cross-reactivity. These probes will be synthesized, characterized, and screened for hybridization efficiency and specificity using spiked groundwater samples to evaluate detection limits, reproducibility, and performance under variable water chemistry conditions (e.g., turbidity, pH, and organic content). Second, we will optimize the assay to detect viable but not proliferating microbial pathogens using spiked ground water.
The anticipated outcome is a novel diagnostic platform that enables rapid, multiplexed detection of diverse pathogens in groundwater. This technology will provide a transformative advance over current methods by shortening detection times, lowering costs, and expanding pathogen coverage. Beyond groundwater monitoring, the platform has potential for broader application in surface water, wastewater epidemiology, and emergency response to waterborne disease outbreaks. By advancing assay technology, this project will contribute to improved water safety, enhanced outbreak preparedness, and stronger public health infrastructure.