COMPARISON OF qPCR-BASED MICROBIAL SOURCE TRACKING DATA TO TRADITIONAL WATER QUALITY MEASUREMENTS IN THE UPPER COHANSEY RIVER WATERSHED *C. D. Phelps,

Slides:



Advertisements
Similar presentations
TMDL Development Mainstem Monongahela River Watershed May 14, 2014.
Advertisements

Patricia Weiss 1, Tiong Gim Aw 2, Joan B. Rose 2 1 School of Public Health, College of Human Medicine, Michigan State University, East Lansing, Michigan,
Anicet R. Blanch Department of Microbiology Microbiología del Agua Relacionada con la Salud (MARS) Intérêts et limites des traceurs de sources microbiennes.
Fecal Colform Bacteria Contamination during Rain Events in Sayler’s Creek, Virginia Blake N. Robertson Senior Honors Research Under the Supervision of.
2009 Water Quality Monitoring Report – Fish Creek Vaughn Hauser, B.Sc. Naomi Parker, B.Sc., BIT, CEPIT.
Recombinant DNA Technology
FECAL SOURCE TRACKING FOR WATER QUALITY C.A. CARSON Food and Agriculture Policy Research Institute Colleges of Agriculture and Veterinary Medicine University.
REAL TIME PCR ………A step forward in medicine
Developments in CSIR's water microbiology laboratory and the introduction of molecular research CSIR NRE.
Identification of E. coli Sources in the Conesus Lake Watershed Using PCR Jason Somarelli Advisor: Dr. Joseph Makarewicz SUNY Brockport Department of Environmental.
Development of Bovine Adenovirus Real Time PCR for Fecal Source Tracking Kelvin Wong, Irene Xagoraraki Abstract Bovine enteric viruses have been suggested.
RESULTS With increasing amounts of Novobiocin there was an obvious decrease in survival of colony forming units of bacteria (Fig. 8). Triclosan was more.
Determination of host-associated bacterial communities In the rhizospheres of maize, acorn squash, and pinto beans.
The Impact of Hurricane Sandy on the Abundance of Coliforms in Tyler Run Michelle Greaver Department of Biological Sciences, York College of Pennsylvania.
Comparison of Regulatory Fecal Indicator Bacteria and Host Specific Genetic qPCR markers in Fecal Matter of Common Sources to Tidal Creeks Aleksandar Dimkovikj.
Presence of Microbial Indicators in Reid Park Wetlands Jepson Sutton Scott Stine SWES 574.
Applied Biosystems 7900HT Fast Real-Time PCR System I. Real-time RT-PCR analysis of siRNA-induced knockdown in mammalian cells (Amit Berson, Mor Hanan.
Point Source POLLUTION: CAUSES AND CONSEQUENCES
What Can You Do With qPCR?
SOURCE TRACKING AND MICROBIAL WATER QUALITY IN THREE FLORIDA SPRINGS Dale Griffin and Mike Gray USGS, St. Petersburg, FL.
Principles and Important Considerations
Lecture 18, Chapter 11 Analysis of transgenic plants part I Mat Halter 3/27/12 Plant Genetics, Breeding and Biotechnology (PLSC 452/552), University of.
Real-Time PCR (Quantitative PCR)
Quantitative PCR Session 2: Overview of qPCR
Analysis of Transgenic Plants. 1.Regeneration on Selective Medium Selectable Marker Gene.
Bacterial Abundance Objective Measure bacterial numbers and mass per unit volume. Note, we are not concerned with identification here. Why do we want to.
Tricia Coakley 1, Gail Brion 2, and Alan Fryar 1 University of Kentucky 1 Department of Earth and Environmental Sciences, 101 Slone Building Lexington,
Generation of Transcription Factor Constructs for Mammalian Transfection Leah Schumerth, Michael Farrell, and Winnifred Bryant Ph. D. Department of Biology.
Real time RT-PCR Quantitating Gene Expression.
West Fork of the White River Stream Restoration Monitoring Dan DeVun Ecological Conservation Organization (501)
Microbial Community Biomarker in Barnegat Bay Evangelina Pena 1, Lora McGuinness 1, Gary Taghon 1, Lee Kerkhof 1 Introduction Efforts to remediate anthropogenic.
NEERS/SNECAFS Joint Meeting, May 9, 2003 Project Author: Kristen Whiting-Grant, Maine Sea Grant Cayce Dalton*, AmeriCorps/Maine Conservation Corps Fred.
TMDLs on the Clearwater River Fecal Coliform Impairment of the Trout Stream Portion of the Clearwater River By Corey Hanson Water Quality Coordinator Red.
Hillsborough River Fecal Coliform BMAP Process Oct. 22, 2008.
Fecal Source Tracking Using Human and Animal DNA U.S. Department of the Interior U.S. Geological Survey Bane Schill- USGS Leetown Science Center.
Rapid detection of pathogenic bacteria in surface water by bacteria universal primer The increase of urban population often results in higher percentage.
 State freshwater fecal coliform criteria  KCEL - Microbial Source Tracking (MST) ‘tool kit’  Summary of findings for Juanita Phase II and Phase III.
E. coli Facts – Beach Monitoring Julie Kinzelman, City of Racine Beach Management Workshop April 14 – 15, 2005, Egg Harbor, WI.
Real-Time Quantitative PCR Basis
Molecular Detection of Karenia spp. in the Mid-Atlantic Kathryn J. Coyne 1, Edward Whereat 1, Muns Farestad 1, Jennifer Torora 1, Lauren Salvitti 1 and.
Richard A. Haugland USEPA, Office of Research and Development, National Exposure Research Laboratory Presented at ORD Produce Expo for Region 5, October.
Molecular Techniques in Microbiology These include 9 techniques (1) Standard polymerase chain reaction Kary Mullis invented the PCR in 1983 (USA)Kary.
Indicator Organisms in Wastewater Treatment Wetlands Jepson Sutton SWES
BacteriALERT: A Program for Monitoring and Real-time Estimation of Indicator Bacteria By Stephen J. Lawrence, Atlanta, Georgia.
112.3 Jessica L. Feeser, M. Elise Lauterbur & Jennifer L. Soong Research Project for Systems Ecology (ENVS 316), Fall ’06 Oberlin College, Oberlin OH BackgroundFindings.
Results of Long-Term Experiments With Conservation Tillage in Austria Introduction On-site and off-site damages of soil erosion cause serious problems.
Diversity and quantification of candidate division SR1 in various anaerobic environments James P. Davis and Mostafa Elshahed Microbiology and Molecular.
Timeline Impaired for turbidity on Minnesota’s list of impaired waters (2004) MPCA must complete a study to determine the total maximum daily load (TMDL)
Richard A. Haugland USEPA, Office of Research and Development, National Exposure Research Laboratory Presented at ORD Product Expo for Region 9, February.
Pine and Mill Creek E. coli Stakeholder Meeting Pine and Mill Creek E. coli Stakeholder Meeting Michigan Department of Environmental Quality, Water Bureau.
Results and Discussion The above graph depicts FC colony plate averages for each sample site. Samples are ordered from upstream to downstream as indicated.
Critique of North Branch of Sunrise River TMDL Nate Topie and Taylor Hoffman.
Findings Is the City of Oberlin a source or a sink for pollutants? Water quality in Plum Creek as a function of urban land cover Jonathan Cummings, Tami.
SPECIFICITY and SENSITIVITY – Performance of Applied qPCR Assays A Bacteria Methanogenic Archaea F FBacteria F FF F Supplementary Figure 1: Specificity.
All Sewers Lead to the Ocean Exploring and Measuring Stormwater Quality SciREN Coast February 12, 2015 Kellen Lauer and Kathleen Onorevole.
Chapter 13: DNA Quantitation.  Determining the amount of DNA is a sample is essential  A narrow concentration range is required for PCR  Must use human-specific.
PRINC E TON School of Engineering and Applied Science Characterizing Mathematical Models for Polymerase Chain Reaction Kinetics Ifunanya Nwogbaga, Henry.
Date of download: 6/24/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Interleukin 6 and Interleukin 8 as Potential Biomarkers.
CORRELATION BETWEEN HYDROLOGICAL, GEOCHEMICAL AND MICROBIOLOGICAL PROCESSES IN GROUNDWATER-STREAM WATER MIXING ZONE Heejung Kim, Seong-Sun Lee, Yunjung.
In poultry houses, ammonia can be harmful to health and performance of both birds and human workers at levels as low as 25 ppm. Poultry litter, excreta.
Biology Program, FDEP Laboratory Evaluation of PMA-qPCR for Quantitative Differentiation of Live Human-associated Bacteroidales for Water Quality Monitoring.
Mulberry River Watershed
Dr. Alice Ortmann University of South Alabama Dauphin Island Sea Lab
QUANTITATIVE ANALYSIS OF AFRICAN SWINE FEVER VIRUS BY DIGITAL PCR
Result Introduction Methods
Raritan Headwaters “Raritan Headwaters is on a mission to protect clean water in the north and south branch region of the Raritan River through science,
COURSE OF MICROBIOLOGY
Supplemental data figure 1.
RealTime-PCR.
Real-Time PCR.
Presentation transcript:

COMPARISON OF qPCR-BASED MICROBIAL SOURCE TRACKING DATA TO TRADITIONAL WATER QUALITY MEASUREMENTS IN THE UPPER COHANSEY RIVER WATERSHED *C. D. Phelps, K. A. Buckley, C. C. Obropta, and L. Y. Young Rutgers, The State University of New Jersey Dept. of Environmental Sciences School of Environmental and Biological Sciences 14 College Farm Rd., Cook Campus New Brunswick, NJ (732) ext ABSTRACT Background: Various methods of microbial source tracking (MST) have been developed for identifying the sources of fecal contamination in the environment. We examine the usefulness of a quantitative PCR-based method targeting host-specific Bacteroides sequences for helping to develop a watershed restoration plan in an impaired watershed (Upper Cohansey River). Methods: An extensive sampling plan involving 10 stations sampled biweekly from June through November with additional events in July, August and September was carried out. Samples were analyzed for pH, DO, temperature, a full nutrient series and fecal coliforms as well as MST. The MST analysis was carried out on surface water grab samples collected in sterile bottles and held at 4˚C until processed. 100 ml of the sample was filtered onto a membrane filter and DNA was extracted from the total filtered biomass. The number of bacteroidetes from all sources (AllBac) along with human-specific (HuBac) and bovine-specific (BoBac) sources was determined by using quantitative, real-time PCR. The qPCR method is a modification of the method developed by Layton et al. (2006). The results from the qPCR analysis are compared to the results from a sanitary survey, fecal coliform monitoring, and water quality measurements. Results: The initial qPCR results show that by using bacteroidetes as a target, fecal contamination can be detected and quantified directly from surface water samples without the use of pre-culturing. The amount of contamination can be determined in terms of total fecal load and the percentage from individual sources (human, bovine and other). These values provide a higher level of discrimination than the other, traditional, measurements of water quality or the sanitary survey. Conclusions: MST based on qPCR can be used to easily identify sources of fecal contamination in watersheds. These results will enable us to expand the use of MST to better prioritize projects and therefore, produce more cost- effective and realistic solutions for microbial contamination in the watershed. Acknowledgements The authors would like to acknowledge the laboratory contributions of Brian Hulme and Ke Shi as well as undergraduate students Thomas Wang and Nicole Lordan. This project was funded in part by the New Jersey Department of Environmental Protection 319(h) Program. Project partners included the Cumberland Salem Conservation District, Rutgers Cooperative Extension of Salem County, and the Rutgers Cooperative Extension Water Resources Program ( CONCLUSIONS  Bacteroidetes from all sources could be readily detected in 100ml surface water samples by using a qPCR assay.  Human and Bovine contributions to fecal contamination could be distinguished from each other.  Pollution sources could be determined by the frequency of detection of specific markers at particular stations over the course of the summer.  Despite the lack of obvious correlations between total Bacteroidetes and fecal coliforms, or any of the other water quality measurements, we were able to gain useful data about the sources and extent of fecal contamination in the watershed. INTRODUCTION  Bacteroidetes  A group of anaerobic, Gram positive bacteria including the genus Bacteroides.  Are good indicator organisms because they do not replicate once released into the environment.  Significantly correlate with the presence of human enteric viruses.  Normal inhabitants of the large intestines of all warm-blooded animals. - Up to cells/gram of feces - Comprise approx. 10% of fecal mass; - 1,000 to 10,000 times more numerous than coliforms  Studies have shown that there are host-specific strains in different animals.  Watershed Characteristics  Freshwater system covering 31 square miles of mixed-use land  73% Agriculture - Includes row crop, sod farms, and field and container nurseries - Livestock, horse and chicken farms  11% Forested Areas and 7% Wetlands  7% Urban  Includes older homes on septic systems  All 10 stations exceed the fecal coliform water quality standard more than 10% of the time.  MST Assay  qPCR used to quantify specific sources of fecal contamination  Based on primers and probes developed by Layton et al. for the detection and quantification of: - All Bacteroidetes (AllBac) - Human-specific Bacteroidetes (HuBac) - Bovine-specific Bacteroidetes (BoBac)References Layton, A., L. McKay, D. Williams, V. Garret, R. Gentry, and G. Sayler Development of Bacteroides 16S rRNA Gene TaqMan-Based, Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution. Applied Environmental Microbiology 72(6): Bernhard, A.E., and K.G. Field A PCR Assay to Discriminate Human and Ruminant Feces on the Basis of Host Differences in Bacteroides – Prevotella Genes Encoding 16S rRNA. Appl. Environ. Microbiol. 66: METHODS Sampling 10 stations were sampled from June through September of In situ measurements of pH, DO, and Temperature made for all samples. Surface water grab samples collected in sterile bottles. Samples held at 4˚C until processing. All samples analyzed for a full nutrient series, TSS, fecal coliforms and Microbial Source Tracking A total of 290 samples processed. MST Assay 100 ml of sample filtered aseptically onto a membrane filter which was cut into quarters using a sterile blade. DNA extracted from total filtered biomass using a DNeasy ® tissue kit (Qiagen). All DNA quantified by spectroscopy and diluted in sterile water to a concentration of 1 µg/ml Used qPCR to measure the number of bacteroidetes present. Total (AllBac) Human (HuBac) Bovine (BoBac) TaqMan ® based assay using Applied Biosystems reagents and standard conditions on an Applied Biosystems 7300 Real-Time PCR system Copy number of each target was calculated by comparison to a standard curve made with plasmids containing human- or bovine-sourced target 16S RNA genes amplified with the primers Bac 32f and Bac 708r (Bernhard and Field, 2000). Brian Hulme (RCE of Monmouth County) filtering a sample. Mike Marandola, Salem RCE, measuring flow in Cohansey. RESULTS  Standards  Clonal libraries of 16S RNA genes generated from PCR of human and bovine feces yielded plasmids specific for HuBac and BoBac primer sets. These plasmids were quantified and used as standards for the qPCR assay.  Dilutions of plasmid DNA provided standard curves which were linear from 10 to 100,000 copies per µL. Figure 2 Standard Curves for quantification of Bacteroidetes: Amplification plot of all three standard curves (a), and the individual standard curves plotting log copy number vs. threshold cycle (Ct) for AllBac (b), Hubac (c), and BoBac (d) primer sets. a b c d  Quantitative Analysis  Bacteroidetes were detectable in samples from all stations at various times.  The number of “human” bacteroidetes was often as high as that of the “total” bacteroidetes and bovine bacteroidetes were rarely detected. 6/28/2006 7/12/2006 Figure 3 Sample Data showing the numbers of bacteroidetes detected by the three primer sets on two days of sampling at all 10 stations. There was 1.59 inches of rain on 6/28 and 0.14 inches on 7/12.  Source Identification  Pollution sources could be determined by the frequency of detection of specific markers at a particular station. Station Copy Number Table 1 Frequency of detection of AllBac, HuBac or BoBac target sequences in samples taken on 10 separate occasions. These data show that certain stations have a higher incidence of contamination with human (C-1, C-2, C-4 and HR1) or bovine (C-3) feces BoBac HuBac AllBac HR1FR1CL-2CL-1C6C5C4C3C2C1 % of Samples Containing Target Sequence Table 2 Comparison of Bacteroidetes measurements by qPCR to other measures of water quality at 3 stations over 5 sampling dates. BD = below detection. These data show the highly variable nature of all of the water quality measures used. C1 C2 C3 C4 C5 C6 CL-1 CL-2 FR1 HR1 Cohansey River Watershed, NJ Figure 1 A map of the Cohansey River watershed showing land use patterns and the surface water quality monitoring locations.