2010 Progress Report: Development and application of a fiber optic array system for detection and enumeration of potentially toxic cyanobacteriaEPA 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.
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.
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.