2009 Progress Report: Development and application of a fiber optic array system for detection and enumeration of potentially toxic cyanobacteria

EPA Grant Number: R833828
Title: Development and application of a fiber optic array system for detection and enumeration of potentially toxic cyanobacteria
Investigators: Anderson, Donald M. , Carmichael, Wayne W
Institution: Woods Hole Oceanographic Institution
EPA Project Officer: Klieforth, Barbara I
Project Period: June 1, 2008 through May 31, 2011 (Extended to May 31, 2013)
Project Period Covered by this Report: June 1, 2009 through May 31,2010
Project Amount: $508,494
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens and Cyanobacteria and Their Toxins in Drinking Water (2007) RFA Text |  Recipients Lists
Research Category: Drinking Water , Water

Objective:

The overall project goal is to adapt and validate a rapid and accurate optical fiber-based technology for cyanoHAB cell detection and enumeration in both laboratory and field settings.  Specific objectives are to: 1) design ribosomal RNA (rRNA) signal and capture probes for the three most important toxic cyanobacteria (Microcystis aeruginosa, Cylindrospermopsis raciborskii, and Anabaena flos-aquae) using published sequences; 2) design and test a second probe pair for each species, to incorporate redundancy into the array; 3) test these probes in the fiber-optic array format and determine detection limits, specificity, and dynamic range; 4) refine hybridization conditions to reduce processing time; 5) develop procedures to analyze multiple cyanoHAB species simultaneously using a single fiber bundle in a multiplexed format and validate it using mixed cultures and spiked and unspiked field samples; 6) work with individuals and agencies responsible for fresh- and brackish water management to determine desired detection limits, precision, new cyanobacteria species for future probe design, and operational characteristics for the assay and instrumentation that would be developed around it; and 7) prepare a detailed protocol for sample handling and processing for use with this method.

Progress Summary:

Major activities during the project period are primarily related to objectives 1-4, and objective 6.  These activities included the identification and testing of existing, published probes that could be adapted to the microarray, as well as the design and testing of probes using DNA sequence data downloaded GenBank.  Probe testing was carried out using fluorescent in situ hybridization (FISH), or "whole cell" analysis, which is conducted routinely in the Anderson laboratory.  Candidate probes were tested against the target and non-target cyanobacteria species we maintain in culture, including multiple strains from each target species as well as closely and distantly related taxa.  Initial testing focused on existing molecular probes, which targeted the small subunit ribosomal RNA gene (SSU rRNA); surprising, all published probes tested either exhibited cross-reactivity or failed to detect one or more of the target strains.  This necessitated the design and testing of new probes, and thus far we have successfully developed molecular probes for C. raciborkii and Microcystis spp.  As toxin production is heterogeneous among Microcystis strains and species, our Microcystis probe was designed to target the genus rather than only detecting M. aeruginosa. Progress on the final probe has been slow due to the complicated and unresolved taxonomy of Anabaena, which is polyphyletic and clusters with Nostoc, Aphanizomenon, and Cylindrospermopsis.  Therefore, any probe designed to detect Anabaena will also detect these taxa; however, we have devised a way to distinguish the Cylindrospermopsis hybridization signal from that of Anabaena, Nostoc, and Aphanizomenon. During microarray data processing, the hybridization signal from the Cylindrospermopsis probe will be subtracted from the hybridization signal obtained from the Anabaena/Nostoc/Aphanizomenon complex probe.  This approach will allow us to effectively distinguish and quantify these taxa, thus circumventing the taxonomic difficulties associated with designing molecular probes for this group. We have identified two candidate probes for the Anabaena/Nostoc/Aphanizomenon complex, and are close to completing the cross-reactivity testing.
 
This year, we also continued work on the fiber optic microarray, primarily focused on testing the Cylindrospermopsis and Microcystis probes in the single probe array. For these experiments, direct hybridization to a labeled oligonucleotide target was first used to test capture probe efficiency; next, sandwich hybridization was performed using a universal oligonucleotide signal probe complementary to a conserved sequence for all three cyanobacteria species. In this assay the signal probe is attached to a fluorophore (typically Cy3 or Cy5) and then compared to the direct assay, in which the synthetic target is attached to the fluorophore. Concentrations of 10 µm to 1 nM were tested. Our preliminary results reveal positive detection of 1 nM of Cylindrospermopsis and Microcystis oligonucleotide targets via direct hybridization within 10 minutes.  No cross reactivity was detected in single arrays when incubated with synthetic non-target samples. Optimization of hybridization conditions is currently underway, after which detection limits for various cell concentrations (5 cells to 5,000 cells) will be determined and detection limits of target 16S rRNA molecules calculated.  
 
In the context of education and capacity building, this project supported Tufts University graduate student Shonda Gaylord for much of 2010 while she worked on this project. Ms. Gaylord will continue to work on this project as part of her Ph.D. studies at Tufts. 
 
Over the past year, we also worked with collaborators to collect field samples from locations in OR, MD, and OH.  These samples were preserved for analysis using the microarray, and subsamples were also preserved for enumeration using light microscopy and fluorescent in situ hybridization (FISH).  Data from the latter two methods will be used to groundtruth data produced by the microarray.  Most recently, we collaborated with Ohio EPA and the Mercer County - Celina City Health Department to collect samples from Grand Lake, OH, during a severe cyanobacteria bloom that occurred during July 2010.  This event caused fish kills and was responsible for at least three dog deaths; health officials are also investigating whether nine people who reported illness after contact with waters in Grand Lake were sickened by toxins in the water. Although the lake was closed to all recreational activities, the city of Celina obtains drinking water from the lake; thus, the occurrence of intense cyanobacteria blooms are clearly cause for concern and the identification of the cyanobacterial community responsible for these blooms is of high priority. Samples were collected from various locations around the lake for community identification; furthermore, paired toxin data will also be available for several of the collection locations to complement our analyses of the cyanobacteria community. 
 
Finally, we are also exploring the feasibility of adapting our probes for use with Luminex Xmap technology, a high-throughput system for nucleic acid detection.  Luminex is used extensively in biomedical research, and has also been successfully employed for the analysis of toxigenic phytoplankton communities.  As with the fiber optic system, this technology utilizes microspheres coupled with capture probes; however, hybridization detection is automated, and uses flow cytometry.  A major advantage of this technology is that the microspheres are internally dyed with two different fluorophores, the combination of which generates up to 100 microsphere sets, effectively permitting the simultaneous detection of 100 species at one time. This system can also be used in tandem with PCR amplification, permitting the inclusion of existing probes that target the genes required for synthesis of microcystin production (mcyD and mcyB) in Microcystis and enabling detection of only the toxin-producing strains.  Due to the low copy number of these genes, these probes cannot be used in the direct hybridization methods employed by FISH and the microarray.  Two demo units are being provided to us by the Luminex Corporation for proof of concept testing, which will commence in September. This technology represents a second powerful and widespread method for rapid enumeration of cyanobacteria in field samples.

Future Activities:

Activities in the upcoming months will focus on completing cross-reactivity testing of candidate Anabaena/Nostoc/Aphanizomenon probes, and adapting the selected probe to the microarray. Setbacks due to difficulties in probe design have delayed progress with the microarray testing, necessitating the postponement of the multiplexing work.  However, upon completing the single probe array work, we will commence experiments with the multiplexed microarray using synthetic targets and cultured cyanobacteria isolates. After completing the multiplexed microarray experiments, analysis of field samples will begin. Proof of concept testing using the Luminex system will also commence in September, and this technology will be evaluated as a second suitable platform for the high throughput detection of toxigenic cyanobacteria.

Journal Articles:

No journal articles submitted with this report: View all 16 publications for this project

Supplemental Keywords:

Health effects, ecological effects, human health, toxics, bacteria, ecosystem, aquatic, environmental chemistry, biology, ecology, genetics, limnology, monitoring, analytical, northeast, central, northwest
, RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Water, Environmental Chemistry, Health Risk Assessment, Environmental Monitoring, Environmental Engineering, Drinking Water, microbial contamination, microbial risk assessment, monitoring, real time analysis, gene microarray assay, aquatic organisms, other - risk assessment, early warning, drinking water contaminants, drinking water system

Progress and Final Reports:

Original Abstract
  • 2008
  • 2010 Progress Report
  • 2011 Progress Report
  • Final Report