setting up a qpcr lab - conference information -...
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Setting Up a qPCR Labg p q
Points to Consider for AccreditationPoints to Consider for Accreditation
Julie Kinzelman, PhDNEMC (Micro Session)
7 August 2012 (1530 1600)7 August 2012 (1530 – 1600)
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Remember QPCR is notMeasuring h h lthe Same Thing as a Culture…
• QPCR differs from traditional culture‐based assays in that it measures all DNA: – live cells– dead cells– non‐culturable cells – free DNA
• Culture assays only measure cells possessing the ability to grow on the selective media you
iare using
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Feasibility StudiesFeasibility Studies• Ability of the method to accurately characterize water qualitywater quality– Will you use the same indicator as your culture‐based assay?
– Is live vs. dead cells important?– Will the assay work with your samples?
• Inhibition, underestimationInhibition, underestimation• Unexplained false negatives w/o inhibition
• Turn‐Around‐TimeWill b bl ll d h l i– Will you be able to collect and process the sample in order to make it “real time”?
• Distance of site to analytical lab– Do you have diurnal variation?
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Choice of Molecular Targetg
• Site Specific Suitability– E. coli or enterococci for fresh water– Enterococci for marine waterB t id ?– Bacteroides?
• Wet chemistry or beadsAssay time– Assay time
– Precision/accuracy• Sample DNA extract ‐ pre‐treatment?• Sample DNA extract ‐ pre‐treatment?
– Crude– Serial DilutionSerial ilution– DNA extraction or purification kits
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Inhibition or UnderestimationInhibition or Underestimation
• Chemical or inorganic constituents in theChemical or inorganic constituents in the sample which prevent reaction from occurring– Humic substances– Humic substances– Non‐point source pollutants
C titi f t t ith b t i i h• Competition for target with bacteria‐rich samples
• Other environmental conditions which result in no detection in the absence of inhibition– i.e. some algal blooms
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Beach Sanitary SurveysBeach Sanitary Surveys
• Guided data gathering at time of sampleGuided data gathering at time of sample collection
• Environmental conditions• Environmental conditions– Wave height, turbidity, recent precipitation, presence of algal biomasspresence of algal biomass
• Can be used to predict site specific inhibition• Can be used in developing predictive models• Can help you make sense of your data!
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Environmental Data Collected – Routine/Daily BSSBSS
• General Beach Conditions • Bather Load– Air temperature– Wind speed/direction– Rainfall
W th diti ( t )
– Total number of people at beach– Swimmers/non‐swimmers
• Potential Pollution Sources– Weather condition (sunny, etc.)– Current speed/direction– Wave Height
• Water Quality
– Sources of discharge• Rivers, outfalls, wetlands, etc.
– Floatables– Amount of debris/litter• Water Quality
– FIB concentrations– Water temperature– Water color/odor
– Amount of debris/litter– Amount of algae
• Stranded on beach• Floating/submerged in waterWater color/odor
– Turbidity (clarity) – Presence of wildlife• Gull counts• Geese, deer, other
Presence of domestic animals– Presence of domestic animals• Dogs, Horses
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Physical Space RequirementsPhysical Space Requirements
• Segregated work spacesg g p– Sample filtration/DNA extraction– Preparation of master mixS l l i– Sample analysis
• Washable surfaces– Non‐porousNon porous– UV disinfection, bleach
• Unidirectional workflow• Dedicated equipment• Dedicated PPE• Proper disposal of DNA waste
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Product Sourcing/CostsProduct Sourcing/Costs
• Consistent product quality• Consistent product quality• Sole suppliers vs. open market• Single lot numbers• Single lot numbers• Wet chemistry vs. beads/kitsM f t d l b d• Manufactured vs. lab‐prepared
• Cost differentialC lt PCR Total analytical costs for qPCR– Culture vs. qPCR Total analytical costs for qPCR
(2 beaches, including QC):
qPCR (E. coli) = $65.00IDEXX (E li) $20 00IDEXX (E. coli) = $20.00
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Staff Training Needsg• Aseptic technique!• Pipetting skillsPipetting skills• Good laboratory practices• Understanding of molecular concepts
– Need comprehensive understanding of assay results• Precision/accuracy• Reproducibilityp y• Academic and hands on
– Learn the basics– Observe a trained professionalObserve a trained professional
• Vendor training• National and regional training opportunities• Train the trainer (referee labs)
• Data review prior to doing it alone
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QA/QCQ /Q• What QC samples should be run?
Method Blank (contamination during sample– Method Blank (contamination during sample filtration)
– Filter Blank (contamination in extraction process)( p )– NTC (reagent contamination)– Calibrator (target quantification check)( g q )– SPC control (extraction and inhibition control)
• Number/Frequency?/ q y• Definition of inhibition
– >3 cycle threshold difference b/w cal‐SPC and3 cycle threshold difference b/w cal SPC and unknown‐SPC (EPA definition)
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QA/QC• Field replicates vs. sample replicates
– Sample replicates = 2 sub‐samples from 1 DNA extraction– Sample replicates = 2 sub‐samples from 1 DNA extraction• Detection in the negative controls
– >35 CT in more than a single NTCg– <45 CT in a third of NTC rxns for a single MM
• Non‐agreement/non‐consensus– Replicate analyses should agree within 1 CT– When is it ok to average replicate CT values?– How do you know what the right answer is?How do you know what the right answer is?
• Reporting out “non‐detects”– Less than half the reciprocal of the dilution factor– What does that mean?
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Sample Handling & StorageSample Handling & Storage• Storing supplies
– Calibrators, Lab prepared cells @ ‐80 °C– Controls, SPC @ 4 – 8 °C– DNA Reagents product dependentDNA Reagents, product dependent– How long?
• Stock salmon DNA was good for 5 years @ 4 °C• Lab prepared cell suspensions were good for 5 years @ ‐80 °C• Lab prepared cell suspensions were good for 5 years @ ‐80 C
• Storing samples– Fresh, analyzed w/i 4 hours of collection– Filters, freeze @ ‐80 °C– DNA extracts, good for 7 days @ 4 °C (up to 1% loss/day)
• Mailing samples and/or reagentsMailing samples and/or reagents– Degradation can occur if temp is not maintained
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Reagents and ControlsReagents and Controls
• ReagentsReagents– Wet Chemistry or bead technologyTroubleshooting contamination– Troubleshooting contamination
• Calibrators and Controls– Lab‐prepared vs. commercial products– Precision (cal curves, replicate sample agreement)– Accuracy (calibrators, SPC)– Reproducibility (b/w run calibrator agreement)
• Inter‐laboratory validation studies
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Comparative Testing (Method Validation)
• Within lab– Can you continue to use the same yprocedures/protocols that you used for culture‐based assays?
• Time of sample collectionp• Composite sampling
– Numerical agreement• Should/could you expect to see this?• Should/could you expect to see this?
– Regulatory action agreement• Will you be as protective to public health and safety?
• Between labs– Inter‐laboratory reproducibility studiesR f l b– Referee labs
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Composite SamplingComposite Sampling• Physical combining of multiple sub‐samples into a single sample– Is it valid to composite?p– Does the composite value fall within the range if individual values?
– Would a composite sample mask a true elevation?
• Increases spatial coverageIncreases spatial coverage• Maintains analytical costs
May be particularly useful when considering qPCR– May be particularly useful when considering qPCR
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Inter‐laboratory Reproducibility StudyInter laboratory Reproducibility Study
• Chosen based on – Level of experience with qPCR– Type of instrument platformyp p– Certification as a water testing laboratory
• Most proficient lab acted as the referee labMost proficient lab acted as the referee lab• Sole source of reagents, calibrators, and controlscontrols
• Same day shipping• Analyze replicate filters within a set timeframe
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Study DesignStudy Design
• Target = E. faecalisTarget E. faecalis• 24 sets of replicate filters from archived surface water samplessurface water samples– 75% unspiked, 25% spiked
• Phase I = analysis of 6 standard curvesPhase I analysis of 6 standard curves• Phase II = analysis of 12 replicate spiked and unspiked filtersunspiked filters
• Phase III = analysis of 12 archived replicate filters from inland waters and Great Lakesfilters from inland waters and Great Lakes
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Results• Standard curves
– 10 100 1000 and 10 000 cells10, 100, 1000, and 10,000 cells– Very good agreement between laboratories– Composite standard curve: R2 = 0.99, PCR efficiency = 101.5%
• Spiked and unspiked filters– No significant difference in DNA recovery as determined by
calculated CCE/100 mL (p = 0 50 0 99)calculated CCE/100 mL (p = 0.50 – 0.99)
• Archived filters– No significant difference b/w participant labsg / p p– Significant difference b/w referee lab and participant labs
• Degraded SPC probes/primers (primarily)DNA l• DNA loss
• Inherent sample variability
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Pilot Implementation – Racine (2011)p ( )
• E. coli– Method 1603 vs. Colilert‐18– Colilert‐18 vs. qPCR– Colilert‐18 vs. Virtual Beach model– qPCR vs. Virtual Beach modelq
• Enterococci– Method 1600 vs EnterolertMethod 1600 vs. Enterolert– Enterolert vs. qPCR
• Relate back to NEEAR study findings• Relate back to NEEAR study findings
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E. coli (Method 1603) and enterococci (Method 1600) were correlated when using culture based enumeration methodscorrelated when using culture‐based enumeration methods
3.5
Log10 Method 1603 vs. Method 1600 (2011)
2 5
3
1603 16001603 LOG10 11600 LOG10 0.65087973 1
2
2.5
LOG10
cfu/100
ml
y = 0.7719x + 0.4609R² = 0.4236
1
1.5
Enterococci, L
0
0.5
0 0.5 1 1.5 2 2.5 3E. coli, LOG10 cfu/100 ml
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Correlation: culture to culture
There is not perfect 3.0
C‐18 vs. 1603, North Beach ‐ AM (2011)
There is not perfect agreement, even between two culture‐ 1.5
2.0
2.5
based methodsy = 0.7283x + 0.3392
R² = 0.72050.5
1.0
0.00.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Anova: Single Factor
SUMMARYGroups Count Sum Average Variance
Colilert‐18 46 4388 95.39130435 102011.8879Method 1603 46 2350 51.08695652 7865.458937
ANOVASource of Variation SS df MS F P‐value F crit
METHOD 1603vs.
Colilert‐18Between Groups 45146.13043 1 45146.13043 0.821755015 0.367088177 3.946875558Within Groups 4944480.609 90 54938.67343
Total 4989626.739 91
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1000
E. coli, Colilert‐18 vs. qPCR (2011)
800
900
C‐18, PM qPCR, PMC‐18, PM 1
600
700
or CCE)
qPCR, PM 0.903933 1
400
500
ts per 100
ml (MPN
C‐18, PM
qPCR, PM
200
300
Unit
0
100
5‐Jul
7‐Jul
9‐Jul
11‐Ju
l
13‐Ju
l
15‐Ju
l
17‐Ju
l
19‐Ju
l
21‐Ju
l
23‐Ju
l
25‐Ju
l
27‐Ju
l
29‐Ju
l
31‐Ju
l
2‐Au
g
4‐Au
g
6‐Au
g
8‐Au
g
10‐Aug
12‐Aug
14‐Aug
16‐Aug
18‐Aug
20‐Aug
22‐Aug
24‐Aug
26‐Aug
28‐Aug
30‐Aug
1‐Sep
Date
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9
E. coli CCE/100 mL vs. Date of Sample (2011)
6
7
8
3
4
5E. coli CCE/100 mL(Log Normalized) NB-AM qPCR Avg. Value
NB-PM qPCR Avg. Value
0
1
2
05 6 7 11 13 14 18 19 20 21 25 26 27 28 1 2 3 4 8 9 10 11
Date of Sample (July 5, 2011 - August 11)
89
E. coli CCE/100 mL vs. Date of Sample (2011)
2345678
E. coli CCE/100 mL(Log Normalized) NB-AM qPCR Avg. Value
NB-PM qPCR Avg. Value
01
5 7 13 18 20 25 27 1 3 8 10Date of Sample (July 5, 2011 - August 11)
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2011 VB Model Pilot Study2011 VB Model Pilot StudyVB Predictions vs. Colilert‐18
Correct, 42/46 91%Incorrect, 4/46 9%Type I, 2/46 4%Type II 2/46 4%Type II, 2/46 4%
VB Predictions vs. qPCR CCECorrect 43/44 98%Correct, 43/44 98%Incorrect, 1/44 2%Type I, 1/44 2%Type II, 0/46 0%yp , /
The predictive model constructed using Virtual Beach accurately estimated E. coliconcentrations enumerated by culture‐based (Colilert‐18, 91%) and qPCR assays (98%).
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Implementation ‐ 2012Implementation 2012• Regulatory monitoring 5 days/week
– Received approval as an ATP indicator/method combination in May 20122 beaches– 2 beaches
– E. coli by qPCR (BioGx Smartbeads™: E. coli and Sketa)Sketa)
– Compare to culture 4 days/week– Compare to model 5 days/weekp y /– Compare E. coli/enterococci by culture (qPCR to follow)
• Archived filters (Fall 2012)
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Data Handling Reality CheckData Handling Reality Check• Replicate analyses do not always agree
– Open, advisory, closed, coin toss (?)• Culture does not always agree with qPCR• Results do not always make sense based on environmental conditionshibi i• Inhibition
• False negatives w/o inhibition (algae blooms?)• Filter blanks or NTC are contaminated• Contamination as a result of visitors/observers
t i PPEnot wearing PPE
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The Way ForwardThe Way Forward• What is the way forward for broader
limplementation?• Further comparative studies
– Know your beach• Years of comparative data
S S S OO• USE THE SANITARY SURVEY TOOL– Correlations b/w inhibition and ambient conditionsVi t l B h• Virtual Beach– Helps make sense of environmental conditions and analytical resultsanalytical results
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Points to Consider• Make sure this analytical method works for you before you start the lab set up process
• Consult with proficient labs• Exchange sample filters• Future needs:
– Sole source or approved QC materials– Accreditation– Laboratory Inspections– Staff Credentials– Proficiency Testing
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A k l dAcknowledgements
• EPA Contract #EP115000072• City of Racine, WI• US EPA Office of Water, NERL, Region 5, , g• Tamara Anan’eva (qPCR Queen of the WI & EPA OW ORISE
Intern)• A long line of dedicated staff and students:
– Jennifer Lavender– Michelle Leittl– Denny Mudd– Jenni Creekmur
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Thank You!