DEVELOPMENT OF RAPID QPCR APPROACHES FOR MEASUREMENT OF E' COLI AND ENTEROCOCCUS IN ENVIRONMENTAL WA

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DEVELOPMENT OF RAPID QPCR APPROACHES FOR
MEASUREMENT OF E. COLI AND ENTEROCOCCUS IN
ENVIRONMENTAL WATERS THE FUTURE FOR ROUTINE
MONITORING?

• Rachel T. Noble, A. Denene Blackwood, and Seth Yu
• National Monitoring Conference
• May 9, 2006
• UNC Chapel Hill Institute of Marine Sciences
• Morehead City, NC

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Bacterial indicator testing

• Routine monitoring in US costs gt10 M USD annually
• Majority of money spent in sampling and analysis
  time
• California alone responsible for more than half
  of the monitoring in the US
• Aim Protect public health
• Impacts Public perception, economy, tourism,
  recreational water usage
• Need Rapid, quantitative methods that can be
  used to accurately manage beaches and shellfish
  harvesting waters

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Routine methods bacterial indicators

• Total coliforms (TC) heterogeneous group composed
  of Escherichia, Citrobacter, Enterobacter,
  Salmonella, Shigella, Yersinia, and Aeromonas
  genera
• Fecal coliforms or E. coli thermotolerant (44.5
  C) subset of TC (FC can include Klebsiella),
  freshwater
• Enterococci, (gt 20 species, e.g. faecalis,
  faecium, casseliflavus, durans, avium,
  gallinarum), marine waters
• Membrane filtration, Chromogenic substrate
  (IDEXX), and Multiple Tube Fermentation
• Require from 18-96 hours for results
• Inaccurate management of recreational and
  shellfish harvesting waters because of delay

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The need for faster results

• Allow accurate management of beaches (keep open
  when clean and close immediately when not safe
  for swimming)
• Faster results better tracking down sources of
  contamination
• Tracking sources down reducing sources (or
  prioritizing reduction of human sources)
  reduction of potential risks to the public

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Immediate future real world example

• 700 AM Beach water sample collected
• 800 AM Return to lab, process using rapid
  method and routine method (MF, MTF, IDEXX)
• 1000 AM
• 1) E. coli gt 400/100 ml and/or Enterococcus
  gt104/100 ml or Return and close beach, sample
  on periodic basis until clean
• 2) Enterococci lt104/100 ml or E. coli lt 400/100
  ml Keep beach open and sample again (or wait
  until next morning to repeat)

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Longer term future

• Same time-scale (or maybe more rapid)
• Measure pathogens instead of indicators (i.e.
  conduct epidemiology studies)
• Real time measurements on deployed systems could
  provide hourly indications of water quality
• Technology applicable to shellfish harvesting
  waters, aquaculture effluents, stormwater runoff,
  NPDES permits etc.

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Criteria for rapid methods

• Time required for result
• Threshold exceedance
• Numerical result
• Accuracy, Variability, Reproducibility
• Portability
• Specificity and usefulness of result for
  mitigation or protecting public health
• Training required
• Data accessibility
• Maintenance required of system (Deployable in
  situ, unattended?)
• Cost (initial buy in, and per sample)
• Equivalent results to classical indicator
  bacteria data/historical data

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Developing Rapid Detection Technologies for
Environmental Waters

• Most currently developed technologies utilize
  sample collection and filtration approaches
  similar to routine methods
• Capture is dependent upon approach
• Detection fluorescence, electrochemical, etc.
• Data transfer and real-time access
• Combination of available applications limited
  only by imagination and
• Noble and Weisberg 2005

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Capture nucleic acid priming

• PCR Based methods
• Transcription mediated amplification (TMA)
• Microarrays
• NASBA
• Highly specific/sensitive
• Based upon known sequence complementarity
• Can be used to type specific types of pathogens

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Rapid microbial detection assays

• Developing technology using QPCR in conjunction
  with Cepheid, Inc.
• QPCR- quantification of E. coli ( 2.0 hr)
• QPCR- quantification of Enterococcus sp. ( 2.0
  hr)
• QRTPCR-quantification of human enteroviruses ( 4
  hr)
• QPCR and QRTPCR- Rapid assays for a wide range of
  other viral pathogens (noroviruses, Vibrio
  vulnificus) and fecal marker bacteria
  (Bacteroides, Enterococcus species)
• Apply technology in all types of water samples
  (estuarine, coastal, freshwater, brackish,
  wastewater, shellfish harvesting waters,
  shellfish harvesting meats)

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QuantitativePCR (or RTPCR)

• Primer/probe set design
• PCR for amplification of DNA
• No gels needed

Cepheid Smart Cycler II

• Increase in fluorescence is directly proportional
  to the amount of target cDNA in sample and
  indicated by cycle threshold

• Standard curve generated by plotting threshold
  cycle vs. log concentration and unknown values
  interpolated

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QPCR Assay Details

• 100 ml through PC filter to capture bacteria
• 0.45 µm pore size with vacuum needs same as for
  MF analysis
• DNA purification (bead beating or full
  extraction)
• Assay features SmartMixTM Beads (all PCR
  reagents) SmartBeadsTM contain primers, probes,
  and internal controls (lyophilized)
• Reduces pipetting steps and errors, interanalyst
  variability, increases quality of standard curves
• Assay also incorporates the use of innovative
  QPCR chemistry developed by DsX Limited
  (Manchester, UK) called SCORPIONS
• Scorpions give lower background, lower LOD
• Licensing fees not prohibitive in cost to WQ
  agencies

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Specificity and Ubiquity
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SCCWRP Study

• June 2005 study to compare rapid detection
  methods for Enterococcus and E. coli to routine
  methods (MF and either Enterolert or
  Colilert-18)
• 3 day study
• 54 blind samples
• Seawater, stormwater runoff, blanks, coastal
  water unseeded and seeded with cultured bacteria
• Verification and speciation of isolates on plates
  and in quantitrays
• QPCR and TMA-based assays both fared well in the
  comparison
• Routine methods roughly 85-90 percent agreement
• QPCR Enterococcus roughly 80 accurate
• QPCR E. coli results 90 accurate
• QPCR had low variability compared to other methods

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Summary of results

• Fully quantitative, wide dynamic range
• LOD for both assays is 1 cell per reaction, for
  assay with DNA extraction step is 10 cells/100 ml
• 90 correct response rate for E. coli and 80-85
  correct response rate for Enterococcus
• Strong correlation to routine methods (r2 values
  range from 0.70 to 0.95 depending upon comparison
  to MF or DS)
• ENT assay captures at least 10 known species of
  Enterococcus (including faecium, faecalis,
  caselliflavus, pseudoavium, gallinarum, etc.)
• Working towards ability to conduct filtration in
  the field
• Internal controls and matrix controls in place
  for quantitative sample analysis
• Currently beta-testing assay at OCSD and
  exploring use in drinking water (E. coli)

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Preliminary results of beta testing at OCSD

• Roughly 40 samples
• Enterococcus and E. coli assay being tested by in
  house WQ personnel
• Results good to date
• Open beaches and storm samples
• Stay tuned!

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AcknowledgementsSteve Weisberg and John
Griffith, SCCWRP Denene Blackwood and Jason
Gregory (UNC Chapel Hill) State of California,
esp. Robin McGraw and Shakoora Azimi-Gaylon United
States Department of Agriculture Cepheid, Inc.
R. Schaller, C. Wilkins, N. Beckwith and S.
Yu Orange County Sanitation District Rich
Haugland, USEPA