7/31/2019 GOMA Presentation Source Molecular USF Delaware
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7/31/2019 GOMA Presentation Source Molecular USF Delaware
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Microbial Source Tracking (MST) at Tower Road Bayside Beach,Rehoboth Bay, Sussex County, Delaware, 2011
Funding Source: EPA Region 3 BEACH Act grant Delaware Environmental Laboratory
Potential sources of contamination(Beach Sanitary Survey) Human Dog Gull
Problem High levels of Enterococcus at an
estuarine inland recreational beach
Goal Determine if Human, Dog or Gull fecal
sources contributed to the highEnterococcus levels
7/31/2019 GOMA Presentation Source Molecular USF Delaware
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Validate the sensitivity of two biomarkers
Human Polyomavirus (HPyVs) Biomarker- USF
Raw untreated wastewater from 3 wastewatertreatment facilities in Sussex County, DE
ResultsSewage tested positive for HPyVs
Gull (Gull-2) Biomarker - USF/EPA
37 fecal samples from herring and/or laughing gullstested individually
Results84% of the samples tested positive for the Catellicoccus gull
marker
Pre-Sampling Analysis
7/31/2019 GOMA Presentation Source Molecular USF Delaware
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Sample Acidification
Samples for HPyVs qPCR analysis requiredacidification (field sample adjusted to 3.5 pH)
Determine if the acidification process would affectthe concentration of the Gull-2 marker
ResultsGull marker detection (CT = Cycle Time) and estimated
concentration lower in acidified samples vs. non-acidifiedsamples
GULL-2 qPCR analysis to be performed on untreated fieldsamples
Pre-Sampling Analysis
7/31/2019 GOMA Presentation Source Molecular USF Delaware
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49 water samples taken through the summer months
Composite sample of 3 locations (taken 3 times per week)
DNREC:
Enterococcus concentrations - Enterolert
Preparation of membrane filters for qPCR analysisqPCR Analysis:
Source Molecular (SM): USF:
Human (HF183 and HPyVs) Human (HPyVs)
Gull Gull
BirdDog
Scope of Work
7/31/2019 GOMA Presentation Source Molecular USF Delaware
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Methods
Membrane Filtration - DNREC0.45 m nitrocellulose filters usedMinimum of 300ml filtered -15 minutes maxFilters were placed in tubes and stored at -80C until
shipped SM- filters rolled and placed into 5 ml MoBio vial with beads USF- filters folded and paced into 2 ml cryogenic vial
7/31/2019 GOMA Presentation Source Molecular USF Delaware
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Extraction and qPCR
Differing extraction kits were used SM - MoBio PowerWater DNA Isolation kit USF - started with MoBio PowerSoil DNA Isolation kit but moved
to PowerWater kit for better results
qPCR assays HPyVs TaqMan (SM and USF) HF183 TaqMan (SM and USF) Gull-2 - TaqMan (SM), SYBR Green (USF)
7/31/2019 GOMA Presentation Source Molecular USF Delaware
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Human Bacteroidetes (HF183) SM 8% of the samples were positive (n=49)Quantification values showed few human specific
genetic copies ( 112 genome equivalents/100ml)
HPyVs - SM and USF Not detected in any sample
Results - Human
7/31/2019 GOMA Presentation Source Molecular USF Delaware
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Source Molecular 100% of the samples were positive (n=48)Using PowerWater DNA Isolation KitQuantification value: 25,365 gene copies/100ml
USF 82% of samples were positive (n=49)
92% positive with PowerWater DNA Isolation Kit
70% positive with PowerSoil DNA Isolation Kit
Quantification values: 13,822 gene copies/100mlConcentrations: SM > USF
(frequency 71%)
Results - Gull
7/31/2019 GOMA Presentation Source Molecular USF Delaware
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HF183 (sub-study)
CT values- No significant correlation(r = 0.431, P = 0.124, n = 14)
Log10
Genome equivalents per 100 ml
Significant correlation at alpha level of 0.10
(r = 0.515, P = 0.059, n = 14)
Gull (results where both labs used PowerWater DNA Isolation Kit)
CTvalues - Significant correlation
(r = 0.561, P = 0.004, n = 25)
Log10Gene copies per 100 ml - Significantcorrelation (r = 0.424, P = 0.034, n = 25)
Correlations Between LabsPearson Correlation
7/31/2019 GOMA Presentation Source Molecular USF Delaware
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HF183 and Enterococcus (sub-study)
HF183 CT values and Enterococcus (Log10 MPN/100 ml)Not Significant
SM (r= 0.015, P = 0.958, n = 14)
USF (r= - 0.213, P = 0.464, n = 14)
HF183 (Log10Genome equivalents/100 ml) and Enterococcus(Log10 MPN/100 ml)
Not significant
SM (r= 0.288, P = 0.319, n = 14)
USF (r= 0.285, P = 0.324, n = 14)
Correlations Between MST Biomarkers and FIBPearson Correlation
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Gull and Enterococcus where both labs used the PowerWater DNA
Isolation Kit
Gull CT values and Enterococcus (Log10 MPN/100 ml)Not Significant SM (r = - 0.312, P = 0.129, n = 25)
Significant USF (r = - 0.406, P = 0.044, n = 25)
Gull (Log10 Gene copies/100 ml) and Enterococcus (Log10MPN/100 ml)
Not Significant
SM (r = 0.285, P = 0.167, n = 25)
USF (r = 0.210, P = 0.314, n = 25)
Correlations Between MST Biomarkers and FIBPearson Correlation
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SM and USF using TaqManCT values - No Significant difference
Log10Gene copies per reaction Significant difference
Log10 Gene copies per 100ml Significant difference
SM using TaqMan and USF using SYBR GreenCT values - Significant difference
Log10 Gene copies per reaction No Significant difference
Log10Gene copies per 100ml No Significant difference
USF using TaqMan and USF using SYBR Green
CT values - Significant difference
Log10 Gene copies per reaction Significant difference
Log10 Gene copies per 100ml Significant difference
Gull DNA Swap Between LabsSource Molecular extracted DNA (n=10); Paired t-test
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SM using TaqMan and USF using SYBR GreenCT values - Significant difference
Log10Gene copies per reaction Significant difference
Log10
Gene copies per 100ml Significant difference
TAKE-HOME MESSAGE:
CT values are not influenced by the error inherent
in constructing a standard curve, and therefore offer a
more direct comparison between results thanestimated gene copies.
Gull DNA Swap Between LabsUSF extracted DNA (n=10); Paired t-test
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Sample processing and storage
Volume and time of water processed Temperature and time period stored
DNA extraction
Extraction kit and equipment Experience of analyst
Potential Contributors to Variation
GeneriteQIAGEN
SIGMA-ALDRICH life technologies
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qPCR reaction
Volume of qPCR reaction Volume of template DNA Master Mix used Thermal cycling conditions qPCR platform and software Primer and Probe sequences Number of replicates tested Standards
Material (eg. Plasmid, genomic DNA)
Range used for standard curve
Potential Contributors to Variation
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Inherent variation
Estimating DNA concentrations in standards Pipetting CT and concentration Log relationship
CT of 35.09 = 365.90 gene copies per 100ml CT of 35.88 = 249.23 gene copies per 100ml 2% RPD between CT equates to a 38% RPD between gene copies/
100ml
Data Interpretation
Determination of outliers (statistical or visual) Selection of the positive CT cutoff How to treat non-detects in statistics: use MDL, MDL, 0
Potential Contributors to Variation
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Biomarkers are not available for all the potential fecalsources in environmental waters.
Cross reactivity for markers with feces from non-target hostspecies must be understood and used in interpretation ofresults.
Is the sampling plan and MST analysis protocol appropriateto answer the question?
What is the scale and budget for the project?
Microbes used for MST markers may not have a directcorrelation to conventional fecal indicator bacteria. Inter-laboratory variability can make comparison of results
challenging.
With close collaboration, labs can identify main sources ofvariability and improve agreement of results.
Concerns and Considerations
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Gull was likely a major contributor to the fecalcontamination detected at the beach.
Human was a possible contributor but occurredsporadically.
MST study purpose was to confirm the visualobservations that gulls contributed to the elevatedlevels of Enterococcus (Delawares water quality
indicator) at this beach. Based on MST results, therewill be no change to management actions at thesite.
Conclusions
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Source Molecular Corporation4985 SW 74th CourtMiami, Florida 33155 USATelephone: (786) 220-0379
Mauricio Larenas
University of South Florida4202 E Fowler Ave, SCA 110
Tampa, FL 33620
Valerie J. Harwood, Ph.D.
and
Katrina V. Gordon, Ph.D.
Delaware Department of NaturalResources & Environmental Control
Edythe Humphries, Ph.D.
Project report available upon request
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