![]() ![]() ![]() Overarching project goals include 1) continuing to develop and optimize PMA-qPCR methods for zoonotic pathogens (including Campylobacter jejuni, Salmonella enterica, E.coli 0157:H7, and Shigella spp.) 2) determining the effectiveness of these methods in differentiating live and dead-cell DNA cells in various environmental matrices 3) validating these methods in a variety of environmental matrices including water, sediment, manure, and algae and 4) evaluating how environmental results can be used to inform QMRAs and gauge potential risks to human health. As part of ongoing research projects, the MI-BaRL develops and optimizes new molecular tools and assays, such as PMA methods for downstream qPCR analysis and quantitative microbial risk assessment (QMRA). The Michigan Bacteriological Research Laboratory (MI-BaRL USGS MI-OH Water Science Center) conducts research on the occurrence, source, abundance, and transport and fate of zoonotic pathogens in a variety of settings including both agricultural and urban environments, rivers, inland lakes, groundwater, and the Great Lakes. Although PMA-qPCR has proven successful in the differentiation of live and dead cells in some matrices, additional method development and validation is required for challenging environmental applications. By correcting standard qPCR assays for detection of only viable pathogens, more accurate risk assessments can be conducted. PMA penetrates the damaged cell walls of dead pathogen cells and inhibits qPCR amplification allowing for a selective assessment of only the live pathogen cells. The propidium monoazide (PMA) approach was introduced recently with the aim of discriminating live-cell DNA from dead-cell DNA in downstream applications such as qPCR. Additionally, accurate estimations of pathogen genes in the environment are critical to understanding and evaluating the effectiveness of wastewater treatment plants, stormwater management, and green infrastructure on reducing human- and wildlife-health risks from combined sewer overflows (CSOs) and wastewater treatment plant effluent, stormwater runoff into neighboring surface waters, and storm drain effluent at recreational waterbodies. The ability to differentiate between live and dead cells would provide a more accurate understanding of risks associated with pathogens delivered from a broad array of sources including animal manure and human biosolids application and wildlife dissemination of zoonotic pathogens. Although there are many benefits to qPCR such as not relying on the ability to culture fastidious microorganisms, one main limitation is the inability to differentiate between live and dead-cell DNA resulting in a potential overestimation of the abundance of pathogen genes. Molecular methods such as quantitative polymerase chain reaction (qPCR) allow scientists the ability to target specific genes allowing for a rapid, sensitive, and cost-effective way to determine the presence and relative abundance of potential pathogen genes in the environment. ![]()
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