Grantee Research Project Results
2002 Progress Report: Detection of Emerging Microbial Contaminants in Source and Finished Drinking Water with DNA Microarrays
EPA Grant Number: R828039Title: Detection of Emerging Microbial Contaminants in Source and Finished Drinking Water with DNA Microarrays
Investigators: Leon, Ricardo De , Straub, Timothy M.
Current Investigators: Straub, Timothy M. , Rochelle, Paul A.
Institution: Metropolitan Water District of Southern California , Pacific Northwest National Laboratory
Current Institution: Pacific Northwest National Laboratory , Metropolitan Water District of Southern California
EPA Project Officer: Hahn, Intaek
Project Period: March 1, 2000 through March 1, 2003
Project Period Covered by this Report: March 1, 2002 through March 1, 2003
Project Amount: $517,818
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
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. The objectives of this research project are to develop and use DNA arrays for natural, turbid, and processed water supplies. Cryptosporidium parvum, E. coli O157:H7, and Helicobacter pylori will serve as model organisms.
Progress Summary:
Work during Year 2 of the project focused on the testing and validation of the C. parvum array. During this time, we were able to obtain and test two genotype II (Iowa and GCH1) and two genotype I (TU502 and UG502) isolates. In addition, our collaborators from the Metropolitan Water District of Southern California provided DNA from a panel of nontarget organisms to further test the specificity of our methods. Both the chemiluminescent (biotin) and direct fluorescence (Cy3) labeled polymerase chain reactions (PCRs) were compared for their ability to provide reliable single nucleotide mismatch discrimination (SNPs) when hybridized to the arrays. We developed statistically robust image and data analysis tools for the analysis of the SNPs.
The results of this work showed that: (1) nontarget organisms failed to amplify using our PCR primers, and furthermore, failed to hybridize to any probes on the microarray; (2) clear differences between hybridization patterns for genotype II and genotype I oocysts were observed; and (3) single nucleotide mismatch discrimination could be achieved for six of the seven positions for both genotype II isolates, all seven positions for UG502, and five of seven positions for TU502.
For the genotype II isolates, SNP discrimination could not be achieved for position 1419 because of potential nucleotide “wobble” at that position. For position 1542, SNP discrimination was temperature dependent for both the Iowa and GCH1 isolate. Increased temperature resulted in statistical discrimination of the SNP, but SNP discrimination for the other positions for the GCH1 isolate was lost due to failed hybridization. SNP discrimination for positions 1479 and 1542 was not possible for the TU502 isolate (genotype I) because of failed hybridization to any probes within the suite of probes for those positions. Better SNP discrimination was achieved when fluorescently labeled PCR products were used. Failure to statistically determine SNPs for the chemiluminescent method was most likely due to the signal generation and amplification processes for developing the biotin label. These processes tended to mask differences in hybridization intensities for perfectly matched probes and mismatched probes.
Work this year also has focused on the development of new generation methods to correlate hybridization results on the microarray with live/dead capability. One method we are investigating is the direct extraction and hybridization of mRNA to the arrays. RNA:DNA hybrids are detected with a specific antibody for these duplexes. Signal is generated by the addition of Cy3 labeled secondary antibodies. The supplier claims that this method can detect as few as 10,000 copies of mRNA. For highly expressed genes such as ribosomal RNA and heat shock proteins, this may translate to cell equivalent sensitivity of 1-10 cells in the sample. If this is true, then this assay, in addition to being potentially suitable for live/dead discrimination, may allow true multiplexed detection of all waterborne pathogens on the array.
Future Activities:
Work during Year 3 will focus on the development of the arrays to detect mRNA signatures for both live/dead discrimination and multiplexed detection of waterborne pathogens. We are forming a collaborative relationship with Dr. Huw V. Smith at the Scottish Parasite Diagnostic Laboratory in Glasgow, Scotland, to test Cryptosporidium isolates from documented outbreaks and regulatory monitoring actions. Concomitant to this collaboration will be the testing of new generation array methods for SNP discovery including the Argonne gel pad arrays (with melting curve analysis) and reverse chemistry labeling. Our collaboration with the Metropolitan Water District of Southern California also will continue, in which we will test their C. parvum isolates and E. coli isolates from their watersheds.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 27 publications | 8 publications in selected types | All 7 journal articles |
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Type | Citation | ||
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Straub TM, Daly DS, Wunshel S, Rochelle PA, DeLeon R, Chandler DP. Genotyping Cryptosporidium parvum with an hsp70 single-nucleotide polymorphism microarray. Applied and Environmental Microbiology 2002;68(4):1817-1826. |
R828039 (2002) R828039 (Final) |
Exit Exit |
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Straub TM, Chandler DP. Towards a unified system for detecting waterborne pathogens. Journal of Microbiological Methods 2003;53(2):185-197. |
R828039 (2002) R828039 (Final) |
Exit Exit |
Supplemental Keywords:
risk assessment, bacteria, signature, effluent, nucleic acids, biology, epidemiology, genetics, pathology, measurement methods., RFA, Scientific Discipline, Health, PHYSICAL ASPECTS, Water, Ecosystem Protection/Environmental Exposure & Risk, Health Risk Assessment, Monitoring/Modeling, Risk Assessments, Environmental Monitoring, Physical Processes, Drinking Water, monitoring, microbial contamination, measurement , microbial risk assessment, microbiological organisms, detection, exposure and effects, other - risk assessment, bacteria monitoring, exposure, treatment, cryptosporidium , human exposure, microorganism, measurement, water quality, assessment technology, DNA microarrays, Helicobacter pyloriRelevant Websites:
None.Progress 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.