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Analytical Methods

Source Tracking Markers. Updated July 2014

MST researchers have been moving away from library-dependent methods to library-independent methods. Library-dependent methods were shown to include high source misclassification rates and higher costs in building large known-source libraries for a particular area (Stewart and others, 2003). A commonly-used library-independent method is the use of host-associated markers. For this method, a targeted genetic sequence of DNA is copied or amplified by the polymerase chain reaction (PCR) into an amount that can be characterized. The use of host-associated markers has gained favor among MST researchers because of improved accuracy and reduced cost and time to perform over library-dependent methods. In addition, PCR methods have the potential to be sensitive, quantitative (qPCR), and automated (Stewart and others, 2003; Santo Domingo and others, 2007).

Host-specific markers have been identified from different groups of fecal-origin bacteria, often from the genus Bacteroides, a bacterium abundant in the gut of warm-blooded animals. The following list describes the different MST markers that have been utilized in the OWML:

  • Two general Bacteroides marker present in most warm-blooded animals (AllBac: Layton and others, 2006; GenBac: Siefring and others, 2008)
  • Two human-associated markers (HF 183: Bernhard and Field, 2000; BacHum: Kildare and others, 2007)
  • A ruminant-associated marker (BoBac; Layton and others, 2006)
  • A gull-associated marker from the species Catellicoccus marimammalium (Gull2; Lu and others, 2008)
  • A dog-associated marker (BacCan: Kildare and others, 2007)
  • A horse/mule-associated marker from Bacteroidales sp. bacteria (HoF597; Dick and others, 2005)
  • A general waterfowl-associated marker (gull, goose, duck, and chicken – GFD; Green and others, 2012)
     

Utilization of MST markers to characterize different sources of fecal contamination has the potential to be a powerful tool in identifying major contributors to water-quality deficiencies (Santo Domingo and others, 2007; Balleste and others, 2010). Given that the application of MST markers is a relatively new tool, broad generalizations should not be made with these results. The potential for regional variability in MST marker results demonstrates the need for local validation in a given study area (Gawler and others, 2007).

The specific uses and limitations of MST results should be noted when applying these tools. While the presence of a source-associated MST marker indicates that fecal contributions have been made by that animal, a negative result for the MST marker does not necessarily signify the absence of fecal contributions from that host animal. Analysis of MST markers by the quantitative polymerase chain reaction (qPCR) allows for an assessment of the level of these markers in a given water sample. However, interpretation of results for quantification will require future studies documenting the relative survival of the markers in environmental waters (Field and Samadpour, 2007), the relative abundance of markers in source fecal material, and ultimately, the public-health significance of marker quantity results. Therefore, at this point in the state of MST science, it is not possible to determine the exact proportion of fecal contamination contributed by the various host-animal sources in a given watershed, water segment, or water sample. Analysis by qPCR does allow for relative comparisons of the MST marker for a specific host animal, i.e. MST marker levels can be compared both spatially and temporally under varying weather and hydrologic conditions. These comparisons may provide the means to identify trends among the fecal source contributions.

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