PCR markers for faecal source tracking
One of the most promising set of tools to emerge from the MST toolbox has been a suite of assays based on the polymerase chain reaction (PCR). PCR assays detect and amplify specific DNA sequences, producing banding patterns and other signals which can be detected and quantified by various means.
One strength of PCR-based methods is that they can distinguish between species of bacteria that are almost impossible to tell apart using phenotypic tests. More importantly, PCR enables the detection of microbes that have so far resisted our attempts to grow them in the laboratory. If these species of enteric microbes are highly host specific, i.e., are resident in only humans or particular animal species, then a PCR assay of the DNA can be a useful MST tool. Here we will discuss some of the PCR markers in the ESR tool box , and the microbial groups they are being applied to. Prices for PCR markers can be found at Costs for faecal discrimination testing. Information on sample collection and storage can be obtained here.
The Bacteroides-Prevotella group
The Bacteroides-Prevotella group of bacteria resides exclusively in the gut of warm-blooded animals. They are strict anaerobes, which means they are highly unlikely to replicate in the environment. They are excreted in higher numbers than the faecal coliforms and therefore a universal PCR marker designed to detect all of the members of the Bacteroides-Prevotella group (termed Total Bacteroidetes) in waterways is a useful indicator of the presence of faecal pollution. Confirmation of a faecal contamination event may have increasing relevance as the debate over environmental replication of traditional bacterial indicators continues.
In addition, differences within the DNA of members of this bacterial group can be exploited, as the existence of different “genetic markers” — segments of DNA — specific to a host animal enabled the design of PCR assays based on DNA markers from these bacteria. The PCR markers are reported as being specific for human, ruminant, dog, horse, or wildfowl pollution (Field and Samadpour, 2007 (external link) ).
At ESR, we have screened faeces from 12 different types of animals using a human specific Bacteroides-Prevotella marker. All but one animal produced a negative result. The exception was the possum, which contained the same genetic marker as humans. We have since developed a Bacteroides-Prevotella marker specific for possums (Devane et al., 2013 (external link) ), which suggests that the group holds promise for distinguishing human from animal sources.
The following table lists the PCR markers used by ESR to identify sources of faecal contamination in marine and freshwater.
|Target group -Assay abbreviation||Microbial target||Reference|
|General faecal marker -GenBac3||Bacteroidales 16S rRNA||Siefring et al. (2008) (external link)|
|Human -BacH||Bacteroidales 16S rRNA||Reischer et al. (2007) (external link)|
|Human -BiADO||Bifidobacterium adolescentis 16S rDNA||Matsuki et al. (2004) (external link)|
|Human -HumM3||Bacteroidales cell wall protein||Shanks et al. (2009) (external link)|
|Ruminant -BacR||Bacteroidales 16S rRNA||Reischer et al. (2006) (external link)|
|Bovine -CowM2||Bovine-specific faecal genetic markers||Shanks et al. (2008) (external link)|
|Sheep -Schill Sheep||Cytochrome b of mitochondrial DNA||Schill and Mathes (2008) (external link)|
|Canine -DogBac||Bacteroidales 16S rRNA||Dick et al. (2005) (external link)|
|Avian -GFD||Avian-specific faecal 16S rRNA||Green et al. (2012) (external link)|
|Avian -E2||Desulfovibrio-like species 16S rRNA||Devane et al. (2007) (external link)|
|Seagull -gull2||Catellicoccus marimammalium 16S rRNA||Ryu et al. (2012) (external link)|
|Possum||Bacteroidales 16S rRNA||Devane et al. (2013) (external link)|
The specificity of above PCR maker assays, as tested against known faecal samples is shown in a table here [PDF, 116 KB]. These assays have all been tested against at least 200 known faecal samples at ESR.
For an example of how we report results generated by PCR markers, please click here [PDF, 211 KB].
Sample collection and storage
If FWA analysis is to be requested, then the same 1 litre of water sample will be sufficient for all PCR and FWA analysis.
Prices for PCR markers can be found at Costs for faecal discrimination testing.
Additional literature on PCR markers can be obtained here.