Project Number: 5347-13000-002-02
Project Type: Specific Cooperative Agreement

Start Date: Sep 30, 2008
End Date: Aug 14, 2010

Objective:
Increased use of recycled wastewater for municipal irrigation heightens the importance of accurate source tracking methods to differentiate sources of fecal contamination in environmental samples. Bacteroides-specific molecular markers and quantitative real-time PCR have been widely used to discriminate human fecal bacteria from other sources, but cross-reactivity of the human Bacteroides primers with canine, swine, and fish feces has been reported. Such cross-reactivity decreases the utility of published primers and probes for source tracking in many recreational areas, as these waters are often impacted by fish and/or dogs. Development of more specific source tracking tools will allow accurate assessment of public health impacts resulting from the use of recycled water for municipal irrigation and/or aquifer recharge.

Approach:
Fecal samples will be collected and Bacteroides DNA will be extracted and amplified using PCR technology. Amplicons will be cloned into plasmids and sequenced to identify uniform nucleotide sequences that can be used as primer and/or probe targets. Candidate primers and probes will be extensively tested for cross-amplification with DNA extracted from a variety of fecal samples (human, bovine, canine, swine, bird, and fish). Finally, the specific primers and probes will be tested in spiked soil and water samples to assess their accuracy in quantification of DNA targets in environmental samples, and to determine the detection limits of the assay. Documents SCA with ASU.