Grantee Research Project Results
2011 Progress Report: Development and application of a fiber optic array system for detection and enumeration of potentially toxic cyanobacteria
EPA Grant Number: R833828Title: 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: Aja, Hayley
Project Period: June 1, 2008 through May 31, 2011 (Extended to May 31, 2013)
Project Period Covered by this Report: June 1, 2011 through May 31,2012
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, 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:
Figure 1. Acarose gel used to ensure 16s rRNA
recovery (microsytis) with 2kb RNA ladder. 2.000
and 1.500 nt ladder placement identified (left) along
with the 23s (2.900 nt) and 16s (1.500) rRNA
sequences (right). Cell counts along with RNA
recovery in ng and total µg are also listed. Red
outlin indicates RNA on gel.
Figure 2. Sandwich hubridizations performed with RNA synthetic target.
Signal probe and target were co-hubridized while incubating on the array.
Prior to hybridization, RNA target and signal probe (at a concentration of
1µm) were heated to 95oC for 5 minutes in hubridization buffer consisting
of SSPE, Denhardt's solution, and formamide. Threshold for a positive
signal was 3* SD of the BG = 4.5 a.u.
Figure 3. Sandwich assay of a 1 in 10 dilution of the RT-PCR product (66 bp
amplicon generated with Cylindrospermopsis-specific capture and signal probe as
primers), Primers were closer in proximity than the capture-universal signal probe
combination, but both are species-specific and cannot be used for the multiplexed
array).
Figure 4. Encoding, bead finding, and hubridization image for approximately 5.00 mycrocytsis cells
in a duplex array. Beads colored in green are microsytstis specific and beads colored in red are
Cylindrospermopsis specific a). Encoding image showing both Micro and Cylindro beads. b). Bead
finding image distringuishing Micro beads from Cylindro Beads. c). Hybridization image in which only
Micro beads (green) should generate a positive signal. Net hybridization signal = 1118 a.u., cross
reactivlty signal from Cylindro beads = 91 a.
Future Activities:
Major activities in the upcoming months will focus on validating the direct labeling approach and testing the multiplexed array using mixed cyanobacteria cultures. Following these experiments, we will test spiked and unspiked field samples, and will compare these results to quantification using light microscopy. Additional activities also will include data analysis and preparation of results for publication.
Journal Articles:
No journal articles submitted with this report: View all 12 publications for this projectSupplemental 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 systemProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.