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A High-Throughput MFQPCR Approach to Simultaneously Quantify FI Bacteria, MST Markers, and Pathogens of Lake Superior Beaches

Author: Elizabeth Hill, University of Minnesota

Abstract: It is widely understood that pathogen contamination of waters used for drinking and recreation can cause illness in humans. Exposure to sewage-contaminated waters is of particular public health concern. While most beach-monitoring programs currently use fecal indicator bacteria (FIB) tests to assess contamination and health risk, pathogen occurrence may be weakly correlated with FIB. Additionally, since FIB are largely ubiquitous in the intestines of most animals, species-specific markers (MST) of fecal contamination are required to elucidate contamination sources in the environment. The overall objective of this project is to simultaneously quantify FIB, MST markers, and pathogens in a single water sample. Using a dead-end hollow-fiber ultrafiltration system, we collected samples from beaches and wastewater effluent in Duluth, MN and Two Harbors, MN. DNA/RNA was extracted, and total bacterial abundance was quantified by qPCR targeting a conserved region of the 16S rRNA gene. During the summer of 2021, we expect to quantify a suite of MST markers, as well as bacterial and viral pathogens using MFQPCR. This MFQPCR platform can simultaneously quantify 96 genes in 96 samples. Upon project completion in Spring 2022, we expect to have a wealth of data, including bacterial/viral abundance, water quality and water chemistry parameters, and weather conditions. These data will be used in further steps of the project to develop a model predicting pathogen concentration from FI and MST marker data, as well as a QMRA workflow estimating pathogen infection risk.