2000 Progress Report: 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 Period Covered by this Report: March 1, 2000 through March 1, 2001
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 project are to develop and use DNA arrays for natural, turbid, and processed water supplies. Cryptosporidium (C.) parvum and/or Helicobacter (H.) pylori will serve as model organisms.
Progress Summary:Research during Year 1 focused on the design, construction, and specificity testing of prototype oligonucleotide microarrays. The C. parvum array consisted of 68 probes targeting a 190 bp region of the hsp70 gene, and was designed to discriminate between human and nonhuman sources of C. parvum and between different species of Cryptosporidium. The H. pylori array consisted of 36 probes targeting the vac and cagA genes, with the intent of discriminating between different genotypes of H. pylori. Polymerase chain reaction (PCR) and microarray hybridization conditions were optimized for C. parvum and H. pylori gene targets, respectively. Several diagnostic nucleotides within the hsp70 gene (positions 1404, 1464, 1479, and 1542) were resolved with single-base mismatch discrimination using a 4-hour, room-temperature hybridization in a 3X SSC buffer. Not all variable bases within the 190 bp region of the hsp70 gene were diagnostic on the microarray. Nevertheless, initial results showed that this simple microarray system unambiguously discriminates between Genotype 1 and Genotype 2 C. parvum parasites. The H. pylori prototype array easily distinguished between cagA + and cagA genotypes, and single-base mismatch discrimination was achieved using a 2-hour hybridization at 50 C in 2X SSC buffer. SNP detection was used to correctly identify and sequence the genotype and genes of ATCC 43504 and ATCC 700392 isolates.
Future Activities:Work during Year 2 will focus on expanding the C. parvum hsp70 array to include downstream diagnostic sequences previously identified through PCR-based studies. The expanded array will be tested against a large panel of Cryptosporidium isolates, including human and nonhuman C. parvum. PCR conditions for the hsp70 gene will be optimized, consistent with their application to drinking water and EPA Method 1622. The prototype bacterial pathogen array will be expanded to include E. coli O157:H7 gene targets, and tested concomitantly against a panel of Helicobacter and E. coli isolates. We anticipate exploring fluorescent detection against the chemiluminescent detection method during Year 2, and the relative efficacy of signal detection in an environmental background. We also anticipate testing the specificity and sensitivity of combined Cryptosporidium, Helicobacter, and E. coli hybridizations as a prelude to the development of a validated "water quality" array.
Relative to our projected research progress (below), we have met all of our Year 1 milestones and are on track to meet our research goals for Year 2 within budget and on schedule.