Grantee Research Project Results
2008 Progress Report: Development and Evaluation of an Innovative System for the Concentration and Quantitative Detection of CCL Pathogens in Drinking Water
EPA Grant Number: R833003Title: Development and Evaluation of an Innovative System for the Concentration and Quantitative Detection of CCL Pathogens in Drinking Water
Investigators: Tzipori, Saul , Walt, David , Zuckermann, Udi
Current Investigators: Tzipori, Saul , Zuckermann, Udi , Walt, David
Institution: Tufts University
EPA Project Officer: Aja, Hayley
Project Period: August 1, 2006 through August 1, 2009 (Extended to July 31, 2011)
Project Period Covered by this Report: August 1, 2007 through August 1,2008
Project Amount: $600,000
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text | Recipients Lists
Research Category: Water , Drinking Water
Objective:
Progress Summary:
Recovery of B. anthracis from 10 L tap water
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samples using a standard HS Core Bowl and a
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manually operated centrifuge.
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Vol. analyzed
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Spike dose
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Percent Recovery
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(L) (# replicates)
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cfu
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cfu
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(mean +/- SD)
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(mean +/- SD)
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10 (4)
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98.5 +/- 0.7
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30.9 +/- 8.9
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10 (10)
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58.4 +/- 9.6
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30.9 +/- 10.8
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10 (10)
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10.3 +/- 0.4
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44.8 +/- 11.9
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Recovery of C. parvum oocysts and B. anthracis from 10 L tap water samples using
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a standard HS Core Bowl and an automated centrifuge.
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Vol. analyzed
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Spike dose
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Percent Recovery
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Spike dose
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Percent recovery
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(L) (# replicates)
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oocysts
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oocysts
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cfu
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cfu
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(mean +/- SD)
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(mean +/- SD)
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(mean +/- SD)
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(mean +/- SD)
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10 (5)
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100 +/- 2.5
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36.0 +/- 15.2
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14.8 +/- 3.8
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90.2 +/- 9.0
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Microbial Contaminant Name
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Information
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Caliciviruses
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Virus (includes Norovirus) causing mild self-limiting gastrointestinal illness
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Campylobacter jejuni
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Bacterium causing mild self-limiting gastrointestinal illness
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Entamoeba histolytica
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Protozoan parasite which can cause short as well as long-lasting gastrointestinal illness
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Escherichia coli (0157)
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Toxin-producing bacterium causing gastrointestinal illness and kidney failure
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Helicobacter pylori
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Bacterium sometimes found in the environment capable of colonizing human gut that can cause ulcers and cancer
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Hepatitis A virus
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Virus that causes a liver disease and jaundice
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Legionella pneumophila
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Bacterium found in the environment including hot water systems causing lung diseases when inhaled
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Naegleria fowleri
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Protozoan parasite found in shallow, warm surface and ground water causing primary amebic meningoencephalitis
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Salmonella enterica
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Bacterium causing mild self-limiting gastrointestinal illness
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Shigella sonnei
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Bacterium causing mild self-limiting gastrointestinal illness and bloody diarrhea
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Vibrio cholerae
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Bacterium found in the environment causing gastrointestinal illness
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T (C)
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time
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95
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5 min
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95
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30 s
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50
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30 s
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35 cycles
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72
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30 s
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72
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7 min
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Future Activities:
We have designed all PCR primers to have similar lengths and annealing temperatures and expect the amplification temperature cycling protocol to be used with only minor modifications. Assymetric PCR has shown initial promise to increase the hybridization signal intensities and will be studied further. Using a higher antisense primer concentration tends to bias the reaction towards the labeled strand, increasing the probability that it will hybridize to the capture probe instead of the complementary product strand in solution. Multiple primers have been designed and purchased (Integrated DNA Technologies, Coralville, IA) with the strategy that those sequences with highest specificity and sensitivity will be used. At least two capture probes per species of interest have been designed to insure redundancy in the event that any sequences exhibit cross reactivity. Before the microarrays arrive, we will focus on validating the primers for the other CCL3 species of interest. Microarray hybridization conditions and stringency washes will then be refined to maximize the specificity of the capture probes.
Supplemental Keywords:
RFA, Scientific Discipline, Water, Environmental Chemistry, Environmental Monitoring, Drinking Water, Environmental Engineering, pathogens, CCL, continuous flow centrifugation, drinking water monitoring, E. Coli, analytical methods, cryptosporidium , contaminant removal, drinking water contaminants, drinking water treatment, Giardia, other - risk management, contaminant candidate listProgress 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.