Development of qPCR Capabilities for Enterococci Analysis
Transcript of Development of qPCR Capabilities for Enterococci Analysis
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Development of qPCR Capabilities Development of qPCR Capabilities for Enterococci Analysisfor Enterococci Analysis
NJ Water Monitoring CouncilJanuary 31, 2007
Diane CalessoUS EPA, Region 2Biological Monitoring Lab2890 Woodbridge AveEdison, NJ 08837-3679723-906-6999
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OverviewOverview
How PCR worksConventional PCR vs qPCROur thermal cycler• Six steps to run qPCR
EPA Programs qPCR could supportEnterococci qPCR StudiesNJ Beach Study 2007
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Target DNA
2n copies per cycle
1st cycle
2nd cycle
3rd cycle
4th cycle
From 1 original copy
20 PCR Cycles 20 PCR Cycles 30 to 40 30 to 40
minutesminutes
235 = 34 BillionCopiesTo Millions or Billions of Copies
PCR 101PCR 101
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Pitfalls of Pitfalls of Conventional PCRConventional PCR
Longer time to resultsPost-amplification processing • Gels• DNA blot
Interpretation subjective• Bands on a gel • Considerable expertise
required
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qPCR qPCR Primer/ProbesPrimer/Probes
SpecificProprietaryExamples• TaqMan®
• Scorpian®
• Amplifluor®
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Applied Biosystems PrismApplied Biosystems Prism™™
vsvsCepheid SmartCyclerCepheid SmartCycler™™
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SmartCyclerSmartCycler™™ ModularityModularity16 Independent ModulesDaisy Chain• up to 6 units• 96 tests
Versatility• run different tests• vary start times• multiplexing• portable
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Step 1. Collect Water SampleStep 1. Collect Water Sample
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Step 2. Filter Water SampleStep 2. Filter Water Sample
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Step 3. Extract DNAStep 3. Extract DNA
SmartMixSmartMixTMTM BeadBead•• Taq enzymeTaq enzyme•• BufferBuffer•• dNTPsdNTPs•• MgClMgCl22
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Step 4. Step 4. Transfer to reaction tubeTransfer to reaction tube
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Step 5. Run Step 5. Run Reaction in Reaction in SmartCyclerSmartCycler™™
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Step 6. ResultsStep 6. Results
Real time• Individual tests• Superimpose for
comparisons
Quantitative Results• Standard curve
developed• cells/ml or cfu/ml
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Programs qPCR Can SupportPrograms qPCR Can Support
Clean Water Act• National Pollutant Discharge Elimination
System (NPDES) • Total Maximum Daily Loads (TMDL)
Safe Drinking Water ActHomeland Security ActBeaches Environmental Assessment and Coastal Health (BEACH) Act
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Enterococci Probe Developed
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Enterococci qPCR Studies
2003 EPA Two Freshwater Beaches
2003 EPA Epidemiological Study Great Lakes
2005 California Rapid Methods Comparison Study
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Results Two Freshwater Beaches
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ResultsEpidemiological Study Great Lakes
-100
1020
3040
5060
7080
90
Sw
imm
ing-
asso
ciat
ed G
I illn
ess
(x 1
000)
1 10 100 1000Enterococcus Geometric Mean
(daily average, QPCRCE/100 ml)
Swimming-associated illness
95% Confidence bound
Adjusted estimates from linear model. Censored values missing
All subjects
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ResultsCA Rapid Method Comparison Study
Agreement between EPA-ORD qPCR method results and reference laboratory culture method results with respect to being above or below the 104 Enterococci/100 ml marine beach water posting criterion
100%39%84%
Agreement among samples above posting criterion by culture
Agreement among samples below posting criterion by culture
Agreement among all samples
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2007 NJ Beach SamplingWeekly sampling• June - August• 44 NJ stations• 26 Long Island stations
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Referenced StudiesComparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis• http://water.rutgers.edu/Source_Tracking/Enterococcus/Compariso
nofEnterococcusmeasurementsinfreshwaterattwobeachesbyqPCRandEPA1600.pdf
Rapidly Measured Indicators of Recreational Water Quality Are Predictive of Swimming-Associated Gastrointestinal Illness• http://http://www.ehponline.org/realfiles/members/2005/8273/8273.
Evaluation of Rapid Microbiological Methods for Measuring Recreational Water Quality; May 2006, Southern California Coastal Water Research Project• ftp://ftp.sccwrp.org/pub/download/PDFs/485_rapid_methods_II.pdf
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LinksThe EMPACT Beaches Project - Results from a Study on Microbiological Monitoring in Recreational WatersEPA 600/R-04/023, August 2005• http://www.epa.gov/nerlcwww/empact.pdf
Microbial Source Tracking Guidance DocumentEPA 600/R-05/064, June 2005• http://www.epa.gov/ord/NRMRL/pubs/600r05064/600r05064.pdf
Rutgers Cooperative Research & ExtensionWater Resources Program• http://water.rutgers.edu/Source_Tracking/MST.htm
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Thank you for your attention
Any questions?