2010 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, 2010 through May 31,2011
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


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, Cylindrospermopsis, and Anabaena) 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 focused on completing probe development and testing (Obj 1); determining the detection limits and dynamic range of molecular probes for the detection of Cylindrospermopsis and Microcystis (Obj 3); and refining the hybridization conditions to maximize probe signal and minimize hybridization time (Obj 4).

During the past year we completed the development of a capture probe for Anabaena, which had been delayed due to taxonomic difficulties associated with distinguishing this genus from Nostoc and Aphanizomenon. Ultimately, we designed a probe that targets all three genera, and recently completed cross-reactivity testing using cyanobacteria cultures of target and non-target taxa. We have begun adapting this probe to the fiber optic array, and soon will be able to move forward with the multiplexing activities described in Obj 5, as well as the analysis of field samples from lakes and ponds (Obj 6).

In addition to completing capture probe development, we continued work with the Microcystis and Cylindrospermopsis probes in the fiber optic array format. Each capture probe was tested with synthetic targets to assess detection limits and establish the linear dynamic range. These tests were first performed with direct hybridization using a labeled capture probe complement to test capture probe efficiency. Next, the capture probes were used in a sandwich hybridization format using an oligonucleotide signal probe complementary to a conserved sequence in both cyanobacteria species. In the sandwich hybridization format, the ribosomal RNA from the target binds to the capture probe, with a subsequent second binding of a signal probe to a different region on the target. In this assay the signal probe was labeled with a Cy3 fluorophore. After stringency washes, the signal from bound target RNA with an attached signal probe, or the labeled synthetic target, was measured. Net signal intensity, defined as the average hybridization signal minus the background signal, was used to distinguish positive from background signals. The threshold for a positive signal was set to three times the standard deviation of the background images.

Probes were tested in this format using synthetic targets and RNA extracts from cultured cells. These tests showed that both capture probes were capable of detecting 100 pM of synthetic target with a 1 hour hybridization in the direct assay (Fig 1). With the sandwich assay the array was shown to have a linear dynamic range spanning three orders of magnitude from 1 x 10-6 to 1 x 10-9 (using Cylindrospermopsis capture probe and Signal Probe 3 as a representative array). No cross-reactivity was detected on single bead arrays when incubated with solutions of synthetic non-complementary targets.

In addition to these activities, we performed a series of experiments designed to maximize hybridization signal and reduce processing time. For the first set of experiments, two types of microspheres (3.1 µm and 4.5 µm) were tested, as well as two concentrations of the Europium dye (0.1 M and 0.01 M) used to encode the microspheres. For this, a duplex array was assembled by encoding two microsphere pools with two different concentrations of Europium dye. Each bead type was imaged with varying exposure times to determine differentiable signal. Overall, signal intensity was maximized using the 4.5 µm beads encoded with 0.1 M Europium dye concentration, and using a 200 ms exposure time (Fig 2, 3).
Next, we tested two different coupling protocols for the DNA capture probes, and two types of hybridization buffers used for the coupling. These experiments showed that a two-step coupling protocol used for the carboxylated beads provided a stronger signal compared with the amine coupling protocol. Two hybridization buffers were also tested to determine which one maximizes DNA-RNA hybridization efficiency. Images taken at 200 ms of light exposure demonstrated that hybridization in Easy Hyb Buffer provided greater signal (i.e., greater hybridization) in comparison to 5x PBS when tested with 1 nM, 500 pM and 100 pM of labeled target (data not shown).
The final set of optimization experiments tested varying concentrations of each capture probe used in the coupling to determine the most efficient bead:DNA ratio. For these experiments, three concentrations (1 µM, 50 µM, 100 µM) of amine-modified DNA capture probe were coupled to carboxylated beads. Each was incubated with 1 nM of synthetic target, after which net fluorescence was determined. While providing a larger standard deviation, 50 µM of DNA target was shown to provide greater signal in comparison to beads functionalized with 100 µM and 1 µM concentrations of the capture probes (Fig 3).
In a parallel effort, we also explored the feasibility of adapting our probes for use with Luminex Xmap technology, a high-throughput system for nucleic acid detection. The Luminex Corporation provided us with a demo Magpix unit, which was used to conduct proof of concept testing using our molecular probes. Unlike the fiber optic microarray, this system can 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 thus enabling detection of only the toxin-producing strains. During this past year we conducted a series of experiments using the Cylindrospermopsis capture probe and cyanobacteria cultures. For this, DNA was extracted and amplified from Cylindrospermopsis cultures comprising varying cell densities. These PCR products were concentrated and tested using the Luminex array. A linear correlation was observed between DNA concentration and hybridization signal (Fig 5); however, variability in DNA concentrations recovered from dilutions with lower cell densities indicated that additional refinement of extraction methodology is needed if the array is to be used in a quantitative manner. However, our preliminary results demonstrated that Luminex may be a second, suitable platform for the rapid and high throughput detection of toxigenic cyanobacteria.
In the context of education and capacity building, this project supported Tufts University graduate student Shonda Gaylord in 2010 while she worked on this project. Ms. Gaylord will continue to work on this project as part of her PhD studies at Tufts. This project also benefited from the participation of Dr. Yunjung Park, a guest investigator in the Anderson Lab, who completed the Anabaena probe development and cross-reactivity testing.
Over the past year, we continued work with collaborators to collect field samples from lakes and ponds with recurrent cyanobacteria blooms. 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).

Future Activities:

Major activities in the upcoming months will focus on adapting the Anabaena probe to the microarray, and on testing the multiplexed array using mixed cyanobacteria cultures and spiked and unspiked field samples. Following these experiments, we will analyze several field samples collected from lakes and ponds with recurring cyanohab blooms, and will compare these results to quantification using light microscopy. Additional activities will also include data analysis and preparation of results for publication.

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, Drinking Water, Environmental Engineering, 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
  • 2009 Progress Report
  • 2011 Progress Report
  • Final Report