Grantee Research Project Results
2007 Progress Report: A Novel Molecular-Based Approach for Broad Detection of Viable Pathogens in Drinking Water
EPA Grant Number: R833011Title: 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: Aja, Hayley
Project Period: July 3, 2006 through July 2, 2009 (Extended to August 31, 2010)
Project Period Covered by this Report: July 3, 2006 through July 2,2007
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
Objective:
The objective of this research is to develop and evaluate a novel, molecular-based approach for broad detection and enumeration of viable pathogens in drinking water.
Progress Summary:
Progress has been made generally according to the project schedule. Work on WGA/WTA method development and evaluation, pre-rRNA RT-PCR, and evaluation of FTA for nucleic acid storage is well underway, according to the schedule designed for the first year of the project.
Work Progress
Work goals for the first year of the project included WGA/WTA method development and evaluation, pre-rRNA RT-PCR, mRNA RT-PCR, filter challenge studies, and evaluation of FTA for storage of DNA/RNA extracts. In general, work has proceeded according to schedule. Progress towards each of these tasks is summarized below.
WGA/WTA:
Preliminary testing of WGA/WTA technologies has been completed. Testing has demonstrated that WGA/WTA can be successfully adapted to improve molecular detection of small quantities of the pathogens selected for this study, including Adenovirus type 41, Echovirus type 13, and Aeromonas hydrophila. WGA/WTA have been applied to dilutions of nucleic acids from individual pathogen types as well as to combinations of these pathogens spiked into a mock environmental water matrix produced by nucleic acid extraction of a surface water sample. Using quantitative PCR (qPCR), increased amounts of nucleic acids were detected in pathogen samples following WGA/WTA. Preparations are being made for experiments that will more precisely evaluate and quantify the ability of WGA/WTA to improve the detection of environmental pathogens. Prior to these experiments, stocks of all pathogens needed to be quantified according to traditional culture-based methods for the purpose of comparison with molecular quantification. Difficulties have been encountered with the cultivation of Adenovirus type 41. CPE produced by the virus is often unclear and inconsistent. If culture-based quantification of Adenovirus cannot be achieved, molecular quantification (of a DNA target) will be substituted. The other study organisms have been successfully quantified by culture methods and are ready for experiments. Small quantities of the study organisms will be combined and spiked into an environmental water sample matrix to determine if the limit of detection using qPCR is improved after pre-amplification with WGA/WTA. There are also plans to evaluate storage of WGA/WTA products on FTA cards.
pre-rRNA RT-PCR:
Initial pre-rRNA RT-PCR assays have been developed for Aeromonas hydrophila and Mycobacterium avium. Preliminary results suggest that these assays are able to detect viability. These methods are currently being evaluated for sensitivity and application to UV and chlorine inactivated organisms. Additionally, experiments are under way to better quantify the stability of Aeromonas in drinking water in order to better evaluate the sensitivity of the pre-rRNA assay.
mRNA RT-PCR:
Work has not yet begun on this aspect of the project. It will begin in year two.
Filter Challenge Studies:
Preliminary preparations for the filter challenge studies have been made. Equipment for the filter apparatus has been ordered and assembled. However, the filter challenge studies have not yet begun due to project staff turnover. Initial recovery experiments are expected to begin in November of 2007. We have made a strategic collaboration with Scientific Methods Inc. to evaluate their disposable capsule filter, which contains the same alumina nanofiber material that the originally proposed Argonide filters contain.
FTA:
Evaluations of Whatman FTA cards as a storage method for nucleic acids are underway. Some modifications of the FTA protocol are necessary so FTA can be applied in this study. Therefore, preliminary investigations of adapted methods are being conducted so that FTA storage experiments can be successfully designed and completed. Downstream processing of FTA cards is designed so that a punch from the FTA card can be directly included in a reaction (e.g., PCR). However, this presents a problem in this study, since a punch would interfere with detection in a qPCR reaction. Therefore, a method of elution from the punch is required. Whatman provides an elution method as an application note. This method has not been completely validated, and its efficiency is unknown. Therefore, some work has been done to determine the recovery efficiency by eluting DNA from a punch to which a known quantity of DNA was applied.
Work has also been done to determine if FTA punches can be successfully used in WGA reactions. The punches have been directly included in WGA reactions, resulting in improved detection of target pathogens. However, applying a sample to an FTA punch prior to WGA does not increase DNA yields as much as adding a sample directly to a WGA reaction. Soon, after all study organisms are successfully quantified and extracted, experiments will be designed to determine the storage abilities of FTA cards (for both nucleic acids and WGA/WTA products) over several months. It will also be determined how FTA can best be incorporated with WGA/WTA to provide better options for storage and downstream detection.
Expenditures to Date
Expenditures to date are on track with those forecasted in the initial budget and accurately reflect the progress made to date.
Results to Date
From preliminary experiments, it seems that WGA is capable of increasing the concentration of target organism DNA in a sample, on average, by greater than 1 log and the total yield of target organism DNA by greater than 2 logs when a pure sample of concentrated DNA is processed. These results are based on WGA of Adenovirus types 2 and 41 and E. coli Famp. Target organism DNA yields appear to decrease somewhat when the organisms are spiked into a large volume of background DNA and when concentrations of the target are very low; however, yields are still appreciably larger when WGA is applied prior to detection. This was seen in experiments with Adenovirus type 2 DNA spiked into an extracted surface water sample. For WTA, RNA concentration is increased about 4 logs, with a total RNA yield of 6 logs greater for Echovirus type 13 RNA. These are preliminary results with a test-version of a WTA kit produced by Sigma. Trials will have to be repeated to see if this amount of amplification can be reproduced under other circumstances, such as in a background matrix of environmental RNA. FTA results show that recovery using the Whatman elution protocol of Aeromonas hydrophila DNA applied to FTA punches is about 10 percent. However, WGA amplification of identical punches yielded DNA concentrations that were approximately half a log greater than in the original sample applied to the punches, which is 3 logs greater than the concentration of the eluted sample. Despite losses from the elution method, WGA may make it feasible to utilize FTA punches for qPCR detection of low copy numbers of target DNA. Preliminary results from the pre-rRNA RT-PCR assays for Aeromonas and MAC show a marked upshift in pre-ribosomal RNA following nutrient enrichment for viable cells.This upshift is observable in a matter of minutes for the rapid growing Aeromonas, and in a matter of a few hours for the slow growing MAC.This suggests these assays are capable of detecting viable organisms.The sensitivity and precision of this assay is currently being evaluated.
Future Activities:
In year two of the project, we plan to:
Continue development and evaluation of the WGA and WTA methods;
Begin mRNA assay development for adenovirus;
Begin nucleic acid extraction work;
Conduct filter performance and recovery experiments; continue FTA evaluation;
Continue evaluation of pre-rRNA methods;
Begin integration of individual methods into a composite method for testing in drinking water.
Work will continue on WGA/WTA and pre-rRNA RT-PCR method development and FTA evaluation. Filtration and nucleic acid extractions studies will begin. Work on mRNA assays for Adenovirus will be conducted. An abstract on the initial WGA/WTA work will be submitted to the 108th General Meeting of ASM.
Journal Articles:
No journal articles submitted with this report: View all 16 publications for this projectSupplemental Keywords:
Whole Genome Amplification, pre-rRNA, Aeromonas, MAC, Adenovirus, Echovirus
, RFA, Scientific Discipline, Water, Environmental Chemistry, Environmental Monitoring, Drinking Water, Environmental Engineering, monitoring, pathogens, biomarkers, drinking water monitoring, polymerase chain reaction, analytical methods
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.