Development and Integration of Quantitative Real-Time PCR Methods for Detection of Mitochondrial DNA and Methanobrevibacter smithii nifH Gene as Novel Microbial Source Tracking Tools |
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| Authors: | Daniel Dancer Craig Baker-Austin James A. Lowther Rachel E. Hartnell David N. Lees L. O. Roberts |
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| Affiliation: | 1. Centre for Environment, Fisheries and Aquaculture Science, European Union Reference Laboratory for Monitoring Bacteriological and Viral Contamination of Bivalve Molluscs, Weymouth, UK;2. Health and Medical Sciences, University of Surrey, Guildford, UK;3. Health and Medical Sciences, University of Surrey, Guildford, UK |
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| Abstract: | A novel microbial source tracking (MST) method based on the detection of human and non-human markers was developed and applied to track the origin of fecal pollution in water systems. Mitochondrial DNA sequences were used to develop new quantitative real-time polymerase chain reaction (qPCR) assays for dog, poultry, and gull. The targets were included as part of a toolbox including human, cow, pig, and sheep assays. A primer and probe set for the detection of the human-specific nifH gene of Methanobrevibacter smithii was also designed as an indicator of human fecal contamination. The assays were tested for specificity and applied to fecal-spiked surface waters and environmental samples collected from two river catchments impacted by sources of human and non-human fecal contamination. The MST methods described were applicable to both spiked waters and environmental samples, and using the two approaches the origin of fecal pollution could be successfully determined in mixed source fecally polluted waters. |
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| Keywords: | Microbial source tracking mitochondrial DNA quantitative real-time polymerase chain reaction (qPCR) |
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