Quantitative PCR-based approach for detection of fecal pollution in water
Type of DegreeDissertation
Agronomy and Soils
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The water quality of many waterways in our state and nation is deteriorating due to point and nonpoint source pollution from human and animal wastes. Accurate identification of contamination sources is essential for developing cost-effective pollution control strategies. Direct detection of host-specific genetic markers by polymerase chain reactions (PCR) has been widely used in identifying sources of fecal contamination in environmental waters. Four studies were conducted and in the first study, experiments were conducted to validate genetic markers associated with deer/elk, goose, dog, and cattle for bacterial source tracking in Alabama. End-point PCR was performed using DNA extracted from 143 fecal samples of target and non-target animal species. The results showed that one of the two cattle markers, the goose markers, as well as the elk/deer-associated markers had acceptable specificity and sensitivity and thus can be used for bacterial source tracking in Alabama. Field validation showed that both humans and Canada geese contributed to fecal pollution in Parkerson Mill Creek. In addition, water samples collected after a significant rainfall event had the highest frequency of host-associated marker detection. In second study, quantitative PCR was used to determine concentrations of host-associated genetic markers. A more practical and reliable approach was developed to determine the limits of detection and quantification for qPCR assays at both analytical and process levels for two cattle-associated genetic markers. Our results indicated the cattle marker, CowM3, had better performance characteristics overall compared with the CowM2 marker. The objective of the third study was to determine if humans and cattle contribute to fecal pollution at a municipal beach in the eastern shore of Mobile Bay, which has been included on Alabama’s 303(d) list due to elevated enterococci concentrations in coastal waters. DNA extracted from water samples was subjected to quantitative PCR targeting general Bacteroidales as well as human- and cattle-associated Bacteroidales. Enterococci were found in all water samples ranging from 2 to 8000 CFU/100 ml. High concentrations of enterococci frequently occurred after significant rainfall events. There was a positive correlation between enterococci and the general Bacteroidales marker. The human-associated marker was detected in 49 out of 101 samples, but only nine samples had concentrations high enough for quantification. Adsorption of DNA by sediment increases the persistence of free DNA in the aquatic environment and thus may cause ambiguities in the identification of recent fecal pollution sources when PCR-based methods are used. In the fourth study, the adsorption and desorption of DNA molecules on both freshwater and marine sediments were quantified using quantitative PCR. Both DNA extracted from raw sewage and purified PCR products were used in the experiment, and their sorption kinetics showed different trends. More DNA was adsorbed on both sediments in stream water than in 5 mM NaCl solution. DNA adsorption on both freshwater and marine sediments was increased in the presence of Mg2+ and Ca2+. Clay content in the sediments was another important factor influencing DNA adsorption capacity. Adsorption data were fitted with equations of Langmuir and Freundlich. The observed DNA adsorption capacity was higher than the maximal capacity estimated from the Langmuir equation, suggesting the presence of multilayer adsorption. Desorption experiments were performed using various solutions and 5–22% of adsorbed DNA was desorbed. The results indicate that more DNA molecules were adsorbed on sediment through ligand bonding than electrostatic bonding. Taken together, quantitative PCR-based bacterial source tracking methods hold great promise for accurate identification of fecal contamination sources in surface waters. Future research is needed to better understand the influence of sediments on the outcome of bacterial source tracking studies.