CICEET Progress Report for the period 9/01/04 Through 3/15/05
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: 9/01/04
Figures |
||
|
||
|
||
|
||
Tables |
||
|
Project Objectives for This 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 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 of detection and capture probes for 16S rDNA/16SrRNA, the malB promoter, the lacZ and uidA genes/mRNA of Escherichia coli., 23S rDNA/23S rRNA of fecal enterococci, 16S rDNA/16S rRNA of Fibrobacter succinogenes, N. gonorrhoeae, the family Vibrionaceae.
2. Optimization of the lysis procedure and nucleic acid isolation techniques.
Progress on Tasks
1. All probes except for the two for Fibrobacter succinogenes and the lacZ gene have been designed.
2. A reproducible procedure has been developed for lysis and isolation of the nucleic acids from bacteria.
3. Difficulties: A difficulty was encountered with a design of a specific 16S rRNA/rDNA probe for E. coli. Although, the proposed for the microarray 16S rRNA probe cross-reacts with Shigella spp., the use of redundant species-specific markers (the malB promoter, and uidA gene) will allow us to differentiate E. coli and Shigella spp. We tested three different techniques for nucleic acid purification techniques, each of them had one or two shortcomings but we are in the processes of optimization of one of them (see 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 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 print microarrays.
2. Preparation of nucleic reference mix containing these microorganisms or their nucleic acids.
Work Plan for Next Reporting Period
From now and through the 2005 summer break, Alexandra Valdes will focus on a design of probes and construction of expression systems for viral nucleic acids. Sherin Mirza will develop references mixes of nucleic acids for bacteria. Both students will work on printing a large batch of microarray slides.
Anticipated Success in Meeting Project Objectives
We are optimistic that Objectives 1 and 2 will be fulfilled by the end of the first year of funding.
Overall Project Timeline Update
Ideally, we planned to start with Objective 3 during year 1. Due to a delay in receiving grant funds, this appears unlikely to happen. There is the likelihood that a no cost extension may be necessary.
Preliminary Data
1. Probes designed so far and examples of some of them printed on test microarrays are shown in Table 1. The probes were selected with approximate melting temperatures around 60° C.
2. In order to isolate nucleic acids from reference cultures and environmental samples we first investigated the procedure described by Bavykin et al. (2001). Our preliminary work with E. coli as well as the work of others published in the literature suggested this technique as adequate for the project. Further testing, however, identified the following problems with the protocol. First, it is not applicable for samples containing large quantities of DNA. In Figure 1, lane 2 contains a wash through after loading nucleic acids from several cultures on a column. Approximately 95-99% of all nucleic acids did not bind to the column. Attempts to use another silica resin (Impaq RG 1080 Sil instead of silicon dioxide) with a higher binding capacity increased the amount of bound nucleic acid but still was not sufficient to retain all nucleic acids (compare panel A and B). It appears that diluting the sample and loading lower quantities of nucleic acids can help to solve this problem. In this case, however, determination of nucleic acid concentrations is needed. Due to contaminating humic acids, nucleic acid concentration may only be determined by the use of fluorescent dye kits with a fluorometer, a time consuming process. The second problem was more serious. The lysis buffer used by Bavikin et al. (2001) contains Triton X-100, a mild detergent, for lysis. Introduction of sodium dodecyl sulfate SDS is not possible since it precipitates with guanidine isothiocyanate also present in the lysis buffer. Therefore, some bacteria are not lysed reproducibly and protein-nucleic acid complexes (marked with letter C in the Figure 1) are not dissolved leading to their irreversible binding to the silica resin and the loss of DNA. Subsequent sonication or bead beating of lysates only partially alleviates the problem, i.e. most protein-DNA complexes still remain intact.
We subsequently tested another technique for nucleic acid separation - a kit from Qiagen. The kit by itself does not come with reagents for initial cell lysis so we employed the proteinase K-SDS lysis method, now routinely used for nucleic acid isolation from environmental samples (e.g. Burtscher et al., 2003; Fera et al., 2004). Since this lysis method includes both SDS and proteinase K, proteins are efficiently removed. The kit works well and it had only two minor problems. First, DNA was washed through the column due to incomplete binding to the resin at the second binding step (see lane 2 in Figure 2). This flaw can be remedied by combining this wash-through fraction with the final preparation of DNA (i.e. lane 5 in Figure 2). The second problem is that the kit is expensive ($10 per sample) and its use is time-consuming to use (6 hours). While we can use the Qiagen kit at the development stages, it is not suitable for our final goal, i.e. development of a simple, fast and inexpensive kit for detection of microbial contaminants.
The third technique that we tested was a combination of a modification of SDS-proteinase K lysis procedure with a hydroxyapatite resin as a nucleic acid separation medium (see Figure 3). Since this lysis method employs SDS and proteinase K, proteins are efficiently removed and DNA-protein complexes are dissolved. So far, the major drawback with the use of this technique is that its lysis step is quite lengthy, up to six hours. We are optimizing incubation times however, during the lysis stage to decrease them to 30-45 minutes, which shortens the whole purification procedure to at most 1.5 hour. We still need to do final adjustments of concentrations and volumes of phosphate buffer to quantitatively separate DNA and RNA but it appears to be achievable.
References Cited
Bavykin, S.G., J. P. Akowski, V. M. Zakhariev, V. E. Barsky, A. N. Perov, and A. D. Mirzabekov 2001. Portable System for Microbial Sample Preparation and Oligonucleotide Microarray Analysis. Appl. Envir. Microbiol. 67: 922-928.
Burtscher, C. and S. Wuertz 2003. Evaluation of the Use of PCR and Reverse Transcriptase PCR for Detection of Pathogenic Bacteria in Biosolids from Anaerobic Digestors and Aerobic Composters. Appl. Envir. Microbiol. 69: 4618-4627.
Fera, M. T.,T. L. Maugeri, C. Gugliandolo, C. Beninati, M. Giannone, E. La Camera, and M. Carbone. 2004. Detection of Arcobacter spp. in the Coastal Environment of the Mediterranean Sea. Appl. Envir. Microbiol. 70: 1271-1276.
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: Riverhead County Center, Suffolk County, New York 11901
Phone number: 631 852-2077
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.
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.
Does this report offer you enough information to adequately address the above questions?
As an initial report, it does. There appear to be some complex issues that need to be resolved; however, it’s apparent that the researchers are aware of, and are addressing the problems.