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Rodent feces can contain dangerous human microbial pathogens that are a serious health hazard to the human population. Currently, there is no known rapid assay to detect rodent fecal contamination. In this study, a rapid PCR-based mitochondrial DNA primer pair was constructed as it enabled the detection of rodent fecal pollution in environmental samples. Environmental samples were all tested using end-point PCR after filtration and then extraction of DNA using the DNeasy Powersoil pro kit. These primers were designed to specifically detect rodent feces that belong to the Rattus Sp. taxonomic group. Rodent samples from various counties in Georgia were obtained and used as positive controls. PCR primers for rat COX-II mitochondria DNA were used to specifically detect rodent fecal DNA from several rat samples. The primers did not cross react with human, dog, cow, deer, chicken, mice, or squirrel fecal DNA. The rodent fecal PCR assay was then successfully used to detect rodent fecal pollution in estuarine water samples. The link between prominent disease-causing pathogens and rodent fecal pollution in heavily populated areas is cause for concern. This study can then aid in rapid detection of rodents, enabling the strategic construction of disease management protocols.

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