Grantee Research Project Results
2008 Progress Report: Detecting Pathogens in Water by Ultrafiltration and Microarray Analysis
EPA Grant Number: R833004Title: Detecting Pathogens in Water by Ultrafiltration and Microarray Analysis
Investigators: Lee, Anthea K. , Rochelle, Paul A. , DeLeon, Ricardo
Institution: Metropolitan Water District of Southern California
EPA Project Officer: Page, Angela
Project Period: July 21, 2006 through July 20, 2009 (Extended to July 20, 2011)
Project Period Covered by this Report: July 21, 2007 through July 20,2008
Project Amount: $594,106
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
Objective:
The ultimate objective of this project is to develop an innovative approach for detecting multiple waterborne bacterial, protozoan, and viral pathogens utilizing large volume ultrafiltration (UF) as a universal pathogen concentration technique, direct extraction of nucleic acids, whole sample genome amplification (WSGA), and hybridization to a multi-pathogen, water quality microarray. The most important aspects in applying molecular pathogen-detection techniques to water samples are the 'front-end' processes of sample concentration and nucleic acid extraction. The most sensitive and specific detection system available will be virtually useless if presented with low concentrations of poor quality DNA. This proposal addresses these critical issues by focusing on methods for concentrating pathogens from large volumes of water and extracting high quality nucleic acids so that the full potential of advanced molecular detection technologies can be realized. The specific objectives of this project are:
Objective 1: Optimize a universal ultrafiltration-based concentration method for all waterborne pathogens.
Objective 2: Improve nucleic acid extraction and recovery from concentrated water samples.
Objective 3: Design a custom microarray to detect waterborne protozoa, bacteria, and viruses.
Objective 4: Confirm infectivity of concentrated pathogens.
Progress Summary:
Year 2 of this project expanded on Objectives 1 and 2, using Salmonella typhimurium and Cryptosporidium parvum as a model organisms to optimize ultrafiltration recovery efficiencies, optimize DNA extraction techniques, and apply whole genome amplification. Ultrafiltration recovery efficiency for Salmonella and Cryptosporidium using the methods described in the 2007 Annual Progress Report were determined (Table 1). The same DNA extraction techniques used for E. coli were successfully applied to S. typhimurium, but not C. parvum. A different DNA extraction technique was used to extract genomic DNA from C. parvum oocysts. Once DNA extraction had been optimized, WGA using mini-REPLI-g® (Qiagen) was applied to S. typhimurium and C. parvum. An E. coli K12 specific microarray was tested on E. coli genomic DNA extracted from ultrafiltration concentrate.
Results to Date:
Ultrafiltration: The ultrafiltration protocol optimized for E. coli (described in the 2007 Annual Progress report) was applied to S. typhimurium and C. parvum oocysts. While not as efficient as for E. coli, genomic DNA could still be extracted and amplified using WGA. S. typhimurium was enumerated in the same manner as E. coli (colony forming units) to determine recovery efficiency. However, as C. parvum is a protozoan parasite that does not grow on agar plates, enumeration methods to determine recovery efficiency were quite different. C. parvum oocysts were isolated using EPA Method 1623 method to separate the oocysts from other products in the ultrafiltration concentrate. Oocysts were subsequently stained with a FITC-conjugated monoclonal antibody specific for Cryptosporidium oocysts (Crypto Cel Reagent, Cellabs Pty Ltd) for enumeration by fluorescent microscopy. Plans to apply ultrafiltration to human adenovirus are in place and initial experiments are scheduled to take place in August, 2008.
Total microbes added to 100L
|
Escherichia coli K12
|
Salmonella typhimurium
|
Cryptosporidium parvum*
|
Human Adenovirus
|
103
|
70.38
|
58.44
|
61
|
In progress
|
104
|
59.37
|
52.25
|
44
|
In progress
|
105
|
70.50
|
64.66
|
In progress
|
In progress
|
Table 1. Summary of ultrafiltration results (% recovery) for E. coli, S. typhimurium and C. parvum. Results shown are an average of 3 independent experiments for E. coli and S. typhimurium for each dosage. C. parvum results are for one experiment. Replicate experiments are ongoing as of this report.
Genomic DNA Extraction from Ultrafiltration Samples: Salmonella samples were processed in the same manner as optimized for E. Coli (2007 progress report) using Chargeswitch © Forensic DNA Purification Kit (Invtrogen), and successfully amplified by WGA. However, genomic DNA from Cryptosporidium oocysts to perform WGA could not be extracted using the same kit. Instead, concentrated oocysts were collected by membrane filtration and then subject to 5 freeze/thaw cycles alternating in a dry ice/ethanol bath and 98o C water bath. Genomic DNA was then extracted using the Mo Bio UltraCleanTM Soil DNA Isolation Kit, following the manufacturer's protocol. WGA was then successful in amplifying C. parvum genomic DNA.
E. coli K12 Microarray: An E. coli K12 microarray was designed and ordered from Combimatrix (2007 Annual Progress Report). Initial hybridization attempts used WGA amplified laboratory grown E. coli genomic as well as E. coli that had undergone ultrafiltration and WGA. Protocols provided by Combimatrx were used, which recommends 1-5 µg of DNA per microarray. Unfortunately after processing and labeling DNA, we seemed to lose a significant quantity, and likely only hybridized less than 0.5 µg of DNA to the microarray. In addition, hybridization conditions were not optimized, thus all microarray spots appeared positive, even factory-built negative controls such as plant genes. Attempts to increase the concentration of WGA-DNA included applying midi-REPLI-g® Kit (Qiagen) on extracted DNA. While this did increase the amount of whole genome amplified DNA (up to 30 µg), processing for the microarray decreased DNA concentrations to less than 1 µg. Optimization of the microarray protocol is ongoing.
Future Activities:
The premise and goals of this project have not changed thus far. Ultrafiltration of Salmonella and Cryptosporidium, while not as efficient as E. coli, were adequate for genomic DNA extraction and WGA. Human adenovirus will be concentrated by ultrafiltration in the next few months, as well as DNA extraction and WGA on the viral genome. The next step for ultrafiltration is to mix the three organisms together, and verify that recovery efficiency is as good as single spikes. Design and production of a custom microarray spanning these 3 organisms will take place during 2009. Testing of the custom microarray and verification of infectivity will take place during the third year of the project. A custom microarray with many water pathogens will also be designed in the third year of the project.
References:
2007 Annual Progress Report STAR grant R833004
United States Environmental Protection Agency Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA, December 2005
Journal Articles:
No journal articles submitted with this report: View all 2 publications for this projectSupplemental Keywords:
RFA, Scientific Discipline, Water, POLLUTANTS/TOXICS, Environmental Chemistry, Biochemistry, Drinking Water, Microorganisms, enteric viruses, aquatic organisms, bacteria, CCL, viruses, drinking water monitoring, microarray analysis, activated carbon, parasites, water quality, contaminant removal, drinking water contaminants, drinking water treatment, ultrafiltrationProgress 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.