CICEET Progress Report for the period 3/15/05 Through 9/15/05

Project Title: Application of inexpensive microarray for assessment of microbiological contaminants in water quality control.
Principal Investigator(s): Andrei Chistoserdov, Josephine Aller and Paul Kemp
Project Start Date:1 September 2004

Figures

Figure 1

Figure 2

Tables

Table 1

Tasks to meet objectives
1. Design probes for Fibrobacter succinogenes and the lac Z genes.
2. Assemble microarrays.
3. Optimization of the lysis procedure and nucleic acid isolation techniques.
4. Validate probe specificity.
5. Preparation of nucleic reference mix containing these microorganisms or their nucleic acids.

Difficulties
Unexpected cross-reactivity was encountered between a number of detector and capture probes (see Figure and text below).

Project Objectives for Next Reporting Period

Objectives
1. Design a set of highly specific probes and compatible capture and detection probes to multiple targets, optimize their performance and assemble microarray.
2. Optimize, test and validate the microarray in artificial mixtures of bacterial and viral contaminants and environmental samples.
3. Create a reference nucleic acid or microorganism mixture, and determine reproducibility and sensitivity of the constructed microarray.

Tasks to Meet Objectives
1. Design probes and generate nucleic acids for and genomic probes for Enteroviridae, Caliciviridae, and Hepatoviridae and include them in printed slides. Redesign several bacterial probes, which hybridize non-specifically.
2. Re-check probe specificity and extend the composition of the reference mix.
3. Complete optimization and validation of the developed microarrays.

Work Plan for Next Reporting Period
We are behind the original schedule, particularly in generation of viral components of reference mixes. Alexandra Valdes has arrived in Lafayette with viral material collected from the Nissequoge River, a ‘lightly’ contaminated (leaching of septic systems from houses bordering river) river along the north shore of Long Island and will work here on a design of probes and construction of expression systems for viral nucleic acids over the next six months. Sherin Mirza will complete optimization and validation of the existing microarrays. She has several references nucleic acids isolated from bacteria, and will augment these nucleic acids with nucleic acids from remaining bacteria strains and viruses. By the end of the reporting period, both students will switch from printing small trail batches of slides to printing a large batch of microarray slides to be taken to the field.

Anticipated Success in Meeting Project Objectives
Although Objectives 1 and 2 have not been fulfilled by the end of the first year of funding, we are close to their completion.

Overall Project Timeline Update
Originally, we planned to start with Objective 3 during year 1. Due to an extended delay in receiving grant funds, and difficulties encountered during this reporting period, this appears unlikely to happen. We will likely request a no-cost extension for the project.

Preliminary Data
1. Design probes for Fibrobacter succinogenes and the lac Z genes.
Probes were designed for 16S rDNA/rRNA of Fibrobacter succinogenes and the lacZ gene from Escherichia coli. The clacZ capture probe binds the lacZ gene of Escherichia spp., Citrobacter freundii, and Klebsiella pneumonia. We also designed and included a probe expected to be specific for Camylobacter jejuni. C. jejuni is specifically associated with guts of birds, particularly poultry. The hipO gene found only in this microorganism has been selected as a target. Probes designed so far and printed on test microarrays are shown in Table 1.

2. Assemble microarrays.
We used microarray slides from Matrix (Hudson, NH) and Acrydite™ modified capture probes. Capture probes were printed on the slides in random triplicates as recommended by Matrix. We use our printer VersArray ChipWriter (Bio Rad, Hurcules, CA) with an SMP3 quill pin. Attempts in using a different type of pin, SMP10, were not successful due to inability of this pin to print a large number of consecutive spots and great variability in spot geometry. Printing protocol was optimized by several printing trials with variable sarcosyl concentrations, humidity, spot arrangements, blotting and pin washes. To check that printer does leave empty spots and there is no carry over between spots, printings were verified by hybridization with the Cy3-(N)₁₀ oligonucleotide.

3. Optimize the lysis procedure and nucleic acid isolation techniques.
Optimization of a technique for isolation and separation of nucleic acid has been completed. The final technique is a combination of a modification of SDS-proteinase K lysis procedure with a hydroxyapatite resin as a nucleic acid separation medium. Since this lysis method employs SDS and proteinase K, proteins are efficiently removed and DNA-protein complexes are dissolved. We used a 100 ml starting volume, since this is the quantity of water typically used in the water quality analysis, which were filtered through 0.2 µM filters. Filers were than used for isolation of nucleic acids. Filtration and lysis is carried out within 1.5 hour. Fractionation of nucleic acid takes approximately 0.5 hour. We also completed an adjustment of concentrations and volumes of phosphate buffer to quantitatively separate DNA and RNA. Figure 1 (panels A, B and C) illustrate efficient separation of DNA and RNA from a Gram positive and a Gram negative bacterium and an environmental sample. Although not needed at this stage of the project, we probably can decrease the processing time to 1 to 1.5 hours.

4. Validate probe specificity.
A series of hybridization experiments were carried out with printed microarrays with and without the target DNA (see Figure 2 as an example). We found that the Cy3-dEcrRNA detection probe binds to the cEfrRNA1 and cNmrDNA capture probe with or without target DNA present. This was unexpected since heteroduplex formation analysis (the Oligoanalyser program from IDT, Technologies, Coralville, IA) showed no substantial homology between the probes. To remedy the problem, the cNmrDNA probe is being redesigned. The cEfrRNA1 probe will simply be removed from the microarray, since we have an alternative cEfrRNA probe. We encountered non-specific binding between the Cy5 - dEcrDNA detection probe and a number of capture probes. As a result, the Cy5- dEcrDNA probe is also being redesigned.

5. Preparation of nucleic reference mix containing these microorganisms or their nucleic acids.
So far, we successfully isolated and separated DNA and RNA from E. coli, E. faecalis and V. vulnificus from pure cultures. DNA and RNA were isolated in large enough quantities and therefore were quantified by UV light absorption. Nucleic acids from these bacteria can also be easily recovered from environmental samples seeded with their culture or in mixes. We have acquired and are in a process of learning the culturing and enumeration techniques for F. succinogenes and C. jejuni. EPA recommended techniques for detection of total coliforms and enterococci are also being adapted for the use in our laboratory to serve as comparative controls for microarray analyses.

Expenditures
Expenditures are in the range anticipated for the work accomplished to date.

End User Advisor Feedback
Name: Dr. Robert Nuzzi,
Organization: Department of Health Services (DHS), Division of Environmental Quality
Location: 360 Yaphank Avenue, Suite 2B, Yaphank, NY 11980
Phone number: 631 852-5806
E-mail: Robert.Nuzzi@suffolkcountyny.gov

1) At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
Potential applications remain those indicated initially, i.e., the ability to differentiate among various pollution sources, which is needed for making informed management decisions to address those sources, to open/close shellfishing areas, bathing beaches, etc., and for the rapid detection of pathogen presence and species for the protection of public health.

2) What, if anything, has changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
Nothing

3) Do you see any key challenges that the researchers may want to address or keep in mind?
They should keep in mind that the methodology, when ultimately developed, must be simple enough, and inexpensive enough, to be used routinely by the agencies charged with protection of public health. Also, the ability for the procedure developed to be used for regulating beaches is inversely proportional to the amount of time required for obtaining results, i.e., the shorter the time span between sample collection and result, the better.

4) Does this report offer you enough information to adequately address the above questions?
The closure of bathing beaches in Suffolk County in the absence of obvious human input continues to suggest non-human influence, and, perhaps, less potential for adverse public health effects. Therefore, the ability to identify, as well as quantify the polluting organisms remains an essential element for management. Research on the pathogenicity of non-human species to humans is required.

5) Other feedback?
The closure of bathing beaches in Suffolk County in the absence of obvious human input continues to suggest non-human influence, and, perhaps, less potential for adverse public health effects. Therefore, the ability to identify, as well as quantify the polluting organisms remains an essential element for management. Research on the pathogenicity of non-human species to humans is required.