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Abstract

Extreme weather events such as hurricanes, tropical storms, and flooding can introduce microbial contaminants into freshwater systems through stormwater runoff, animal waste, and damaged or overwhelmed wastewater infrastructure. Rapid detection of waterborne pathogens is important for environmental monitoring and public health response, especially in coastal regions vulnerable to hurricane-related flooding. This study developed and refined a polymerase chain reaction (PCR)-based environmental DNA detection technique to identify aquatic pathogens in freshwater systems. Water samples were collected from four freshwater sites, including three man-made ponds and one drainage ditch, during the Fall 2025 and Spring 2026 semesters. Target organisms include Giardia, Cyclospora, Cryptosporidium, Shigella, Salmonella, Escherichia coli, and Hookworm. PCR amplification products were analyzed using agarose gel electrophoresis and compared to expected DNA fragment sizes for each primer set. Amplification products corresponding to expected target fragment sizes were observed for six of the seven targeted organisms, while Cryptosporidium did not produce detectable amplification products during the study period. Site 1, Turtle Pond, demonstrated the greatest diversity and frequency of target amplification events, suggesting that some freshwater sites may require closer monitoring. Giardia, Cyclospora, Shigella, and Hookworm were the most consistently amplified targets and became the focus of further detection technique refinement. Temperature gradient PCR, separation of Giardia primer sets, and reduction of PCR cycle number improved band clarity and helped strengthen the detection method. Overall, this study demonstrates that conventional PCR can serve as an accessible molecular tool for detecting aquatic pathogen DNA in environmental water samples and provides a foundation for future water quality surveillance after flooding and hurricane events.

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