Detection of Emerging Microbial Contaminants in Source and Finished Drinking Water with DNA MicroarraysEPA Grant Number: R828039
Title: Detection of Emerging Microbial Contaminants in Source and Finished Drinking Water with DNA Microarrays
Investigators: Chandler, Darrell P. , Leon, Ricardo De
Current Investigators: Straub, Timothy M. , Rochelle, Paul A.
Institution: Battelle Memorial Institute, Pacific Northwest Division , Metropolitan Water District of Southern California
EPA Project Officer: Nolt-Helms, Cynthia
Project Period: March 1, 2000 through March 1, 2003
Project Amount: $517,818
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Description:DNA microarrays represent a potentially significant technology and analytical technique for the simultaneous detection of multiple pathogens in a single water sample, with the ability to incorporate live/dead discrimination via mRNA analysis. However, microarrays have not been applied to environmental samples (in general), and commercial array companies are focused solely on the high-throughput genotyping and drug discovery markets. We propose to overcome the limitations of culture and PCR-based analytical techniques through the development and use of DNA arrays specifically for natural, turbid and processed water supplies, using Cryptosporidium parvum and/or Helicobacter pylori as model organisms. Critical research questions and hypotheses include:
- Can state-of-the-art microarrays be successfully applied for the genetic analysis of relatively low-biomass source and finished water samples? What are the lower detection limits of DNA arrays in a water quality context? How are microarray detection limits and specificity affected by relatively large nucleic acid targets? Do co-extracted humic acids and environmental contaminants affect detection sensitivity or fluorescent readout strategies?
- Does surface charge modulation on tunable surfaces significantly enhance detection sensitivity and oligonucleotide specificity on microarrays? What are the practical consequences of tunable surface films relative to probe design, hybridization conditions, and detection sensitivity?
- What array formats and internal controls are required for quantitative analysis of C. parvum and/or H. pylori targets in raw water samples? How does microarray sensitivity, specificity and quantification ability compare to a standard IFA technique?
Approach:Major tasks associated with this objective include 1) probe selection and design, 2) array fabrication and specificity testing in various genetic and chemical backgrounds (source, finished waters), 3) characterization of possible interactions between environmental samples (e.g. humic acids, particulates) and fluorescence labeling/readout methods, and 4) evaluation of microarray quantitative ability relative to currently accepted immunofluorescence assays (IFA).
Expected Results:DNA microarrays represent a potentially significant technology for routine monitoring and epidemiological studies of waterborne disease that may include live/dead discrimination (via mRNA analysis) and simultaneous, quantitative detection of multiple pathogens in a single sample. However, microarrays have not been applied to environmental samples (in general), and commercial array companies are focused solely on the high-throughput genotyping and drug discovery markets. Consequently, this project will represent the first application of advanced microarray technology to water quality analysis, and will provide:
- Fundamental information on how factors such as hybridization buffers, ionic strength, temperature, nucleic acid concentration (total and target-specific), target size, microarray surface derivatization and charge density, probe spacing and density, probe size, and labeling strategy affect hybridization efficiency and sensitivity of detecting microbial pathogens in environmental samples (source and finished water).
- Novel information on the utility of surface modification chemistries (i.e., tunable microarrays) to increase hybridization detection sensitivity for water quality samples.
- Novel information on the applicability of commercial DNA microarrays for the analysis of turbid natural source and finished waters. This research will represent the first time that microarrays are optimized and used as an assessment technology for analysis of environmental samples.
- Development of the first quantitative DNA microarray specifically for water quality monitoring applications and the analysis of C. parvum.