Grantee Research Project Results

2007 Progress Report: Detecting Pathogens in Water by Ultrafiltration and Microarray Analysis

EPA Grant Number: R833004
Title: 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, 2006 through July 20, 2007
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 1 of this project focused on Objectives 1 and 2, using E. coli K12 as a model organism to optimize ultrafiltration recovery efficiencies, optimize DNA extraction techniques, and compare whole genome amplification kits. Despite a 7 month delay in starting the project (due to a contractual error), we are on track to completing objectives 1 and 2 using the bacterial model organism, E. coli K12. Ultrafiltration recovery was improved to an average of 72.2%. Four genomic DNA extraction kits were compared and 4 whole genome amplification kits were compared. From these tests, we have chosen the optimum method for concentrating E. coli K12, extracting genomic DNA, and applying whole genome amplification.

Results to Date

Ultrafiltration. Ultrafiltration of 100 L of was optimized for E. coli K12. We saw improvement from an average of 33.14% recovery to 72.2% recovery. The vast improvement was a primarily a result of changing the elution buffer from a solution of 0.1M Tris-HCl and 0.01% Tween 20, to a solution of 0.1% sodium polyphosphate, 0.01% Tween 80, and 0.001% Y-30 antifoam as published in (1). Addition of blocking agents such as sodium hexametaphosphate and fetal bovine serum had no additional experimental or cost-effective benefits in our hands.

Whole Genome Amplification. Four whole genome amplification (WGA) kits were tested on 1 ng, 10 ng, or 30 ng of purified E. coli K12 genomic DNA. WGA yields for 10 ng of DNA are shown in Table 1. DNA concentration was measured using Qubit Quantitation Fluorometer™ (Invitrogen). Qiagen REPLI-g^® and GE Healthcare Genomiphi™ V2 kits use Multiple Strand Displacement (MDA) technology which involves binding of random hexamers followed by strand displacement synthesis by Phi29 DNA polymerase. Sigma GenomePlex^® Kit generates a fragmented library of genomic DNA with attached universal primers, followed by PCR amplification. DOP-PCR by Roche uses degenerate oligonucleotide primers for PCR amplification with no preprocessing.

WGA products were also visualized on an agarose gel to determine the quality of the amplified DNA (Figure 1). Equal concentrations (0.25 μg) of DNA were loaded onto a 0.8% agarose gel. As expected, GenomePlex^® Kit resulted in smaller fragments due to the generation of a fragmented library, while both MDA kits had a variety of larger fragments. Interestingly, DOP PCR resulted in many distinct bands, indicating that perhaps the amplification method was quite biased. Although both kits using MDA technology performed quite well, we have chosen to use REPLI-g^® (Qiagen) for ease of use and slightly higher average yields.

Table 1. Comparison of WGA Kits. 10 ng of genomic DNA from laboratory-grown E. coli K12 was amplified using the whole genome amplification kits indicated. *Starting material of 10 ng genomic DNA as published in manufacturer’s manuals. **Results from 2 independent experiments.

Kit	Expected Yield* (μg/mL)	Actual Yield** (μg/mL)
REPLI-g® Ultrafast Mini (Qiagen)	350-500	357 644
Illustra™ Genomiphi™ V2 (GE Healthcare)	200-350	317 214
GenomePlex® Complete (Sigma)	40-93	30 None detected
DOP-PCR (Roche)	Not specified	9 5

Figure 1. Visualization of DNA Quality. Each lane of the 0.8% agarose gels were loaded with 0.25 μg of the WGA product from the indicated kit. 1 kb ladder (New England Biolabs) was used as reference.

Genomic DNA Extraction from Ultrafiltration Samples. Ultrafiltration often concentrates minerals and other small particles, like humic acids, in addition to the microbial target. Some of these particles can interfere with downstream molecular methods like DNA amplification. Four genomic extraction kits were tested on E. coli K12 that had been concentrated by ultrafiltration. All 4 genomic DNA extracts were visually brown, indicating some sort of mineral or small particle contamination. Therefore we used the PowerClean™ DNA Clean-Up Kit (MoBio Laboratories, Inc) to remove the contaminants. WGA was then performed using the REPLI-g^® Kit (Qiagen) on both the cleaned and uncleaned DNA samples. The concentration of DNA was measured using Qubit Quantitation Fluorometer™ (Invitrogen), although it is unclear if the brown color of “uncleaned” samples interfered with the readings to give artificially high concentrations, or if the “cleaned” DNA truly reduced DNA concentration. The Invitrogen Chargeswitch^® Forensic DNA Purification Kit performed the best with regards to ease of use, efficiency of DNA recovery, and quality and quantity of DNA amplified by WGA (Table 2).

Table 2. Concentration of Total Genomic DNA (“Uncleaned DNA” Column), Followed by PowerClean™ DNA Clean-Up Kit (MoBio Laboratories, Inc) (“Cleaned DNA” Column). WGA was carried on both “uncleaned” and “cleaned” DNA for comparison (Qiagen). Table lists the result of one experiment. 1 mL of E. coli K12 ultrafiltration concentrate (final cfu 2.8x107) was used for each kit, except IT 1-2-3™ FLOW (Idaho Technology) which used 5 mLs of concentrate per the manufacturer’s instructions.

DNA extraction Kit	Uncleaned DNA (ng/μL)	WGA on uncleaned DNA (ng/μL)	Cleaned DNA (ng/μL)	WGA on cleaned DNA (ng/μL)
DNeasy® (Qiagen)	0.285	40.15	0.0429	11.3
Dynabeads® DNA DIRECT™ Universal (Invitrogen, formerly Dynal)	18.85	182	0.114	14.7
Chargeswitch® Forensic DNA Purification Kit (Invitrogen)	2.46	122	0.36	151.5
IT 1-2-3™ FLOW (Idaho Technology)	None detected	None detected	None detected	None detected

E. coli K12 Microarray. An E. coli K12 microarray has been designed and ordered from Combimatrix. The array consists of 40 base pair probes designed every 800 base pairs along the genome for a total of ~5800 probes. Cognate mismatches for each probe and factory standard controls are included in each array. This microarray will be used to determine the robustness and bias of WGA on concentrated E. coli K12 samples. If the amplification of the genomic DNA is unbiased, we will expect complete hybridization to the microarray, excluding cognate mismatches and negative controls.

References:

Hill VR, et al. Applied and Environmental Microbiology 2007;73:4218.

Future Activities:

The premise and goals of this project has not changed thus far. Tests using E. coli K12 will conclude by the end of 2007. We will then move on to optimizing concentration, DNA extraction, and WGA for virus and parasite models (Human adenovirus and Cryptosporidium, respectively), as well as Salmonella. Concomitantly, design and production of a custom microarray will take place during 2008. Testing of the custom microarray and verification of infectivity will take place during the third year of the project.

Journal Articles:

No journal articles submitted with this report: View all 2 publications for this project

Supplemental Keywords:

RFA, Scientific Discipline, Water, POLLUTANTS/TOXICS, Environmental Chemistry, Biochemistry, Microorganisms, Drinking Water, microarray analysis, viruses, ultrafiltration, water quality, contaminant candidate list, contaminant removal, parasites, activated carbon, aquatic organisms, drinking water contaminants, drinking water treatment, enteric viruses, drinking water monitoring, CCL

Progress and Final Reports:

Original Abstract

2008 Progress Report

2009 Progress Report

2010

Final