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: 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: 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:

Major activities during the project period included determining the detection limits and dynamic range of molecular probes for the detection of Cylindrospermopsis, Microcystis, and Anabaena (Obj 3), refining the hybridization conditions to maximize probe signal and minimize hybridization time (Obj 4), and working with the multiplexed array for the simultaneous detection of the three target taxa.

Previously, direct hybridization experiments were carried out using labeled capture probe complements to determine the efficiency of each capture probe. Probes were subsequently used in a sandwich hybridization assay (SHA) format using an oligonucleotide signal probe complementary to a conserved sequence (synthetic target). Work carried out during the current project year focused on testing the probes in the SHA format using cultures of each target species: Cylindrospermopsis (strain LB2897), Microcystis (strain LE-3), and Anabaena (B1444). For these experiments, RNA was extracted and quantified, and gel electrophoresis was performed to confirm RNA recovery of both the 23s and 16s rRNA. RNA extraction was most successful for Microcystis, with yields in the µg range. In addition to testing the intact RNA, RNA was fragmented (60-200 bp in length) in an effort to reduce the steric hindrance and stability of the secondary structure. RNA fragmentation was performed using different target concentrations, fragmenting solution volumes, and incubation times, and tested using the array. However, neither intact nor fragmented RNA was detected in the sandwich assay format.

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 95_oC 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.

In an effort to identify the factors that potentially could contribute to the lack of signal, synthetic DNA and RNA were tested on the array. The array was capable of detecting 100 pM of synthetic target within 30 minutes (Fig 2). In addition, RT-PCR amplicons were tested (using the capture and signal probes as primers capture probe complement was used) on the array, and again this generated positive signal (Fig 3). Based on these data, and a review of the literature, the lack of signal from the extracted RNA could potentially be due to 16s rRNA secondary structure and/or proximal placement of the universal signal probe in relationship to the capture probes.

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).

As an alternative approach, the RNA currently is being directly labeled using the Universal Linkage System (ULS) kit from Kreatech. This step eliminates the secondary binding of the universal signal probes and still allows for fragmentation of the RNA as previously reported. This approach looks promising, albeit with increased incubation times. Generally, detection of rRNA can take anywhere between 2 hrs to overnight incubations depending on the system used and quality of the RNA, probes, etc. Despite the rapid hybridization and signal generated with synthetic targets (60 bases in length), the actual 16s rRNA requires longer incubation times. Initial experiments using a duplex microarray assay with Microcystis RNA (about 5 000 cells), Microcystis beads (green), and Cylindrospermopsis beads (red) provided positive signal with labeled RNA with a 6 hour incubation (Fig 4).

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.

Using Microcystis cells, work is currently underway to determine the shortest incubation time (12 hrs, 6 hrs, 3 hrs or 1 hr) that still provides positive detectable signal while generating a low cross reactivity signal. Thus far, the array is able to detect approximately 1 500 Microcystis cells/mL within 3 hrs. Subsequent experiments will test the multiplexed bead array response in the presence of only one target analyte to assess cross-reactivity, followed by experiments testing all three target analytes to observe simultaneous detection of all three 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. The extensive experimentation that Ms. Gaylord is carrying out with the microarray will comprise part of her Ph.D. thesis at Tufts. This project also benefited from the previous participation of Dr. Yunjung Park, a guest investigator in the Anderson Lab, who completed the Anabaena probe development and cross-reactivity testing.

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 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, Health Risk Assessment, Environmental Chemistry, Drinking Water, Environmental Engineering, Environmental Monitoring, microbial contamination, gene microarray assay, early warning, microbial risk assessment, monitoring, aquatic organisms, drinking water system, drinking water contaminants, other - risk assessment, cyanobacteria

Progress and Final Reports:

Original Abstract

The 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.