Journal of Emerging Scholarship

High bacterial levels in recreational bodies of water can be a risk to human health, and significant effort and funding are invested in monitoring fecal indicator bacteria (FIB) levels. Despite the health risk, methods commonly utilized to determine bacterial concentrations provide no information about the source of contamination. This study assesses the feasibility of utilizing quantitative polymerase chain reactions (qPCR) to perform microbial source tracking (MST) that will identify the source of fecal contamination in rivers and streams in Anne Arundel County. Human and canine fecal bacterial DNA samples were analyzed using primer sets previously reported to target genes frequently identified in host-specific bacterial species. Primers specific for esp, encoding the enterococcal surface protein often associated with human fecal bacteria in clinical settings, and primers specific for the Bacteroides 16s rRNA genes, either specific to bacterial genomes from canine or human sources, were utilized. Quantitative poly- merase chain reaction (qPCR) analysis demonstrated that, while the primer sets successfully amplified target sequences, there was some amplification of non-target sequences within the target host, and some amplification of genes in samples from non-target hosts, such as amplification of sequences in dog bacterial DNA by human bacterial-specific primers. Gel electrophoresis and DNA sequencing of sample qPCR products were conducted and confirmed that target genes were amplified, although the identity of some of the off-target products remains to be determined. Primers reported to target sequences in bacteria from dog feces showed higher specificity, but still resulted in some off-target amplification. On-going work includes optimizing assay conditions and primer sequences to increase specificity and reducing potential sources of reaction contamination which may be contributing to some off-target results.

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