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OWML: QA/QC

Quality Assurance/Quality Control Manual: Ohio Water Microbiology Laboratory
 

Molecular methods for microbial source tracking markers and cyanobacteria
(Note: Appendices for molecular  methods are available upon request)

Molecular methods for microbial source tracking (MST) markers and cyanobacteria used by the OWML target genetic sequences in bacteria using quantitative polymerase chain reaction (qPCR). Polymerase chain reaction is a technology in which DNA from a targeted gene is amplified, generating millions of copies, in order to determine if the targeted gene is present. For quantitative polymerase chain reaction (qPCR), a fluorescent signal is used to quantify the amount of targeted gene relative to a known quantity positive control.

Microbial source tracking (MST) is a term used for the process of identifying the source of fecal contamination in the environment. Microbial source tracking using molecular markers is carried out by detecting genetic sequences in the DNA of fecal-origin bacteria that are specific to the host species that produced the feces. Host-associated molecular markers have been identified based on the theory that the physiology in the gut of the host animal (e.g. diet, temperature, antibiotic treatment, etc.) is unique from one animal to another. These unique conditions select for unique subsets of microorganisms in the gut. Host-associated 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 OWML has adopted the capability to analyze for the MST marker assays listed in Table 8.

Table 8:  Microbial source tracking (MST) marker qPCR assays

Marker Source Targeted bacterium Reference
AllBac General Bacteroides Layton and others, 2006
GenBac General Bacteroides Siefring and others, 2008
HF183 Human Bacteroides Seurinck and others, 2005
BacHum Human Bacteroides Kildare and others, 2007
BoBac Ruminant Bacteroides Layton and others, 2006
Gull2 Gull Catellicoccus marimammalium Sinigalliano and others, 2010
BacCan Dog Bacteroides Kildare and others, 2007
HoF597 Horse/Mule Bacteroides Dick and others, 2005
GFD General waterfowl Helicobacter Green and others, 2012

Toxic freshwater cyanobacterial blooms are of concern in many parts of the world because of their effects on drinking water, water-based recreation, and watershed ecology. Microcystins are one of the most frequently detected hepatotoxins in freshwaters and are generally produced by strains of the genera Microcystis, Planktothrix and Anabaena (Rantala and others, 2006). For toxin production to occur, the microcystin synthetase genes (mcy) must be present in the genome of toxic strains. Known microcystin-producing genera include both toxic strains (with the mcy genes) and nontoxic strains (without the mcy genes), which can be differentiated only by molecular detection methods qPCR. The OWML has developed the capability to analyze samples by several cyanobacteria molecular assays using qPCR (Table 9). The DNA-based qPCR assays reveal the presence of toxin genes (irrespective of whether they are actively producing toxin). The RNA-based assays, using reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), can detect microcystin-producing cyanobacteria that are actively expressing the toxin genes (Sipari et al., 2010).

Table 9:  Cyanobacterial assays for freshwater studies (toxic strains refer to the ability to produce microcystin)

Assay Description Reference
Cyanobacteria, general DNA assay, targets several cyanobacterial genera Rinta-Kanto and others, 2005
Microcystis spp. DNA assay, targets toxic and non-toxic strains of Microcystis Rinta-Kanto and others, 2005
Anabaena spp. DNA assay, targets toxic and non-toxic strains of Anabaena Doblin and others, 2007
General mcyE DNA assay, targets toxic cyanobacteria Rantala and others, 2004
Microsystis and Anabaena mcyE DNA and RNA assays, targets toxin genes specific to Microcystis and Anabaena Sipari and others, 2010
Planktothrix mcyE DNA and RNA assays, targets toxin genes specific to Planktothrix Rantala and others, 2006

Because they are both bacterial targets, the steps for processing and analyzing samples by qPCR for MST markers are similar to those for cyanobacteria. Studies incorporating MST often include analysis of known-source fecal samples or sediment samples in addition to water samples; sample collection and filtration steps are split into separate protocols for these two different matrix types (solid material and liquid material). 

1.      Sample collection and initial processing. 

a.       Collection of water samples for MST and cyanobacterial analyses is done following the same USGS protocol as is used for indicator bacteria (Myers and others, 2007).  One difference in sample collection is that the bottles used to collect the samples should be acid-treated to remove all DNA, RNA, or DNA/RNA degrading substances (Appendix D1). Appendix D1a is a benchsheet that is used for any sample type being processed for MST.

b.      Collection and initial processing of known-source fecal samples for MST markers is done following the protocol in Appendix D2 and using the fecal sample collection field form (Appendix D2a).  

c.       Collection and initial processing of sediment samples for MST markers is done following the protocol in Appendix D2.

 

2.      DNA and RNA extraction and purification.

a.       DNA extraction and purification of a sample for MST and cyanobacteria is done using the GeneRite DNA-EZ kit as described in Appendix D3.

b.      RNA extraction and purification of a sample for cyanobacteria is done using the MoBio PowerPlant RNA isolation kit with DNase as described in Appendix D7.

c.       Extraction information for each sample is recorded on the “filtration and extraction” benchsheet (Appendix D1a).

d.       The extraction and purification process is done in sample batches which are recorded on a benchsheet (Appendix D3a).

 

3.      qPCR for MST markers and cyanobacteria.

a.       Detection of MST markers and cyanobacteria can be done by following Appendix D4.

b.      Details and benchsheets for MST assays can be found in Appendix D4a.

c.       Details and benchsheets for cyanobacterial DNA assays can be found in Appendix D4b.

d.      Details and benchsheets for cyanobacterial RNA assays can be found in Appendix D4c.

 

4.      Positive control development.  Positive controls are used to establish known quantity standard curves which are used to quantify unknown samples and ensure the qPCR reaction was performed properly. 

a.       E. coli plasmids containing the target sequence for MST and cyanobacterial assays are generated and quantified as described in Appendix D5 for use as positive controls.

b.      A plasmid-based positive control benchsheet can be found in Appendix D5a.

5.      Standard curves.  Successful establishment of positive controls will allow for subsequent development of a standard curve which will be used to quantify unknown samples. 

a.       Standard curve development of plasmid-based positive controls can be found in Appendix D5.

b.      Standard curve information for each qPCR run is recorded on the assay-specific benchsheet (Appendices D4a-D4d).

 

6.      Data interpretation.

a.       A protocol for the handling of qPCR data, assessment of matrix inhibition, and recording of final results can be found in Appendix D6.

b.      Data templates for water, sediment, and fecal-source samples can be found in Micro/Current Molecular Protocols.

   

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