A Novel Molecular-Based Approach for Broad Detection of Viable Pathogens in Drinking WaterEPA Grant Number: R833011
Title: A Novel Molecular-Based Approach for Broad Detection of Viable Pathogens in Drinking Water
Investigators: Meschke, John Scott , Cangelosi, Gerard A.
Institution: University of Washington , Seattle Biomedical Research Institute
EPA Project Officer: Klieforth, Barbara I
Project Period: July 3, 2006 through July 2, 2009 (Extended to August 31, 2010)
Project Amount: $597,987
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
The overall objective of the proposed project is to develop and evaluate a novel, molecular-based approach for broad detection and enumeration of viable pathogens in drinking water. The specific objectives of the proposed project are: 1) to develop and evaluate cutting edge filtration and microfluidic methods for pathogen concentration and purification from drinking water; 2) to develop a two-step detection approach, based on general amplification of the extracted metagenome and metatranscriptome followed by pathogen specific detection by viable RT-PCR methods; and 3) to evaluate the performance of the developed method for a variety of spiked and unspiked samples from regional water utilities.
Organisms selected for this study include several CCL organisms (Mycobacterium avium, Aeromonas hydrophilla, Echovirus, and Adenovirus). Filtration using novel positively charged filter media will be evaluated in seeded studies for concentration and recovery of organisms in comparison to hollow fiber ultrafiltration. Also, novel microfluidic methods will be evaluated in seeded studies against commercial spin column kits for nucleic acid extraction. For the first step in the detection approach, novel whole genome and whole transcriptome amplification methods will be adapted and applied to total nucleic acid extracts from water concentrates and short-term enrichments to achieve a limited initial amplification. As a second step, fluorescent RT-PCR methods will be developed targeting specific sequences in target organisms indicative of viability (e.g. precursor ribosomal RNA for bacteria, replicative forms for Echovirus, or mRNA for Adenovirus). The composite method (consisting of the best performing concentration and purification methods with the combined two-step detection method) will be evaluated on seeded and unseeded samples obtained from regional utilities.
The proposed research will develop a novel approach for concentration, purification, and detection of viable pathogens in drinking water that is broadly applicable across pathogen classes (e.g. DNA and RNA viruses, and gram negative and acid fast bacteria). The overall method developed in this study will improve the ability to assess risk associated with microbial contamination of drinking water by providing a rapid, sensitive, and quantitative method for the detection of known and emerging pathogens in drinking water. Furthermore, the proposed study will offer a novel approach for both prospective and retrospective investigation of waterborne infectious disease outbreaks.