Advancing Methods for the Application of Human Mitochondrial DNA in Fecal Source Tracking
by
Kevin Zhu
Advisor(s):
Dr. Joe Brown (CEE & UNC at Chapel Hill)
Committee Members:
Dr. Konstantinos T. Konstantinidis (CEE), Dr. Xing Xie (CEE), Dr. Christine Stauber (GSU), and Dr. Joel Kostka (BIO)
Date & Time: July 2, 2021 3:00 PM
Location: https://bluejeans.com/927228461/1179
Complete announcement, with abstract, is attached
Mitochondrial DNA (mtDNA) is shed in feces and can be an unambiguous host-specific indicator of the origin of fecal contamination in the environment. Human mtDNA offers advantages over microbial fecal source tracking (FST) markers in environmental monitoring because it is highly specific to humans and does not vary across geographies or populations whereas variability in the human gut microbiome suggests limitations in the application of microbial markers. Methods for the quantification of mtDNA, its association with microbial hazards in environmental media, and its persistence in the environment compared with microbial markers are important unknowns that constrain the application of mtDNA as a monitoring tool.
We developed a novel human mtDNA FST assay, hCYTB484, on a droplet digital polymerase chain reaction (ddPCR) platform. Using human feces from the US, Mozambique, and Bangladesh as well as sewage and animal feces from the US, we demonstrated that hCYTB484 is highly human-specific and highly sensitive in human feces from geographically distant regions. We also investigated concentrations of hCYTB484 in feces from individuals with and without norovirus and diarrhea, finding that hCYTB484 concentrations were elevated in feces from individuals with both norovirus and diarrhea compared to those without. These results highlight human mtDNA as a potential candidate for evaluation for association with risk of illness through exposure to waters contaminated with fecal waste. Finally, we simulated fecal pollution events using human feces and sewage diluted into freshwater, quantifying human mtDNA and microbial FST markers over 14 days. We found that differential persistence emerged between mtDNA and microbial markers, suggesting that the detection/non-detection and quantitative relationships between these different markers may provide information about the source and age of the fecal pollution. These results demonstrate how human mtDNA markers may contribute to the FST field by complementing the shortcomings of current widely used microbial markers.