goma presentation source molecular usf delaware

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


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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


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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


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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


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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


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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)


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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


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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


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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


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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



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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



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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

[email protected]

University of South Florida4202 E Fowler Ave, SCA 110

Tampa, FL 33620

Valerie J. Harwood, Ph.D.

[email protected]

and

Katrina V. Gordon, Ph.D.

[email protected]

Delaware Department of NaturalResources & Environmental Control

Edythe Humphries, Ph.D.

[email protected]

Project report available upon request

goma presentation source molecular usf delaware

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