E. Coli is a commonly used indicator of sewage contamination at public swimming beaches.
In 2005, closing and advisory days at U.S. ocean, bay, and Great Lakes beaches topped 20,000. This project resulted in an assay that can be adapted to test for a variety of disease-causing organisms.
How’s the Water?
New DNA hybridization assay provides sensitive, species-specific way to test beach water quality.
Every year, thousands of beaches around the U.S. are closed due to contamination by microbes such as fecal bacteria or harmful algae. Current testing methods involving cell culture and microscopy tools can take days to show results. Because levels of disease-causing microbes change much more rapidly than that, contaminated beaches often remain open when they should be closed, and clean beaches are often closed after the threat of contamination has washed away. To balance the protection of human and economic health, water quality managers need accurate, quick, and cost-effective approaches to pinpoint the type, level, and source of microbial contamination.
With funding from CICEET, researchers from the University of Miami and NOAA’s National Atlantic Oceanic and Meteorologic Laboratories have developed a DNA hybridization microplate assay that quickly and accurately identifies multiple species of toxic plankton and fecal indicator bacteria.
Researchers tested two groups of organisms: toxic dinoflagellates that cause “red tide,” and bacteria that indicate the presence of sewage. They created molecular probes that would bind to the DNA of these microorganisms, and attached the probes to the wells of microplates. DNA from a water sample was then placed in each well. If there was a match, a chemical reaction caused a yellow color to indicate the presence of that organism in the water sample.
The red tide assay successfully detected “very low” levels of Karenia brevis, a red tide organism common in Florida coastal waters. The sewage-indicating assay detected Escherichia coli, total coliforms, the Bacteroides fragilis group (anaerobic bacteria found in humans and animals), and Bacteroides distasonis (associated with the human gut). The Bacteroides assays are a promising way to distinguish between human and nonhuman sewage contamination in coastal waters.
This project resulted in a DNA hybridization microplate assay that provides sensitive, species-specific molecular analysis in a convenient, adaptable, and relatively inexpensive format. Water quality managers can adapt this technique to the molecular probes of their choice, or it can be used as an introduction to molecular methods for educational purposes.
The assay allows for easy sample storage and helps differentiate between human and non-human fecal pollution sources. It provides immediate visual results, is more specific and convenient than a series of species-specific PCR reactions, and is faster, easier, and less expensive than cloning. It is simple to add and adapt probes to the assay. The technique conserves the amount of genomic DNA utilized, which can be critical to certain applications.
The assay process and a video tutorial are available online: http://ciceet.unh.edu/project_extras/microplate_assay
Dr. Jack Fell
University of Miami
Dr. Kelly Goodwin
National Atlantic Oceanic and Meteorologic Laboratories
Multichannel Handheld Sensor for Microbial Contaminants [Progress Report]
Acrobat Towed Instrument Platform: Data Collection, Calibration, and Interpolation/Graphic Visualization [Progress Report]
Microbial Source Tracking in Two Southern Maine Watersheds [Project Brief]
Microplate Assay Development for Detecting Microbial Contaminants [Project Brief]
Application of an Inexpensive Microarray for Assessment of Microbiological Contaminants in Water Quality Control [Progress Report]
F + RNA Coliphages as Source Tracking Viral Indicators of Fecal Contamination [Progress Report]
Microbial source tracking Using F-specific Coliphages and Quantitative PCR [Progress Report]
New Autonomous Technology for Monitoring Microbial Indicators of Fecal Contamination in Coastal Waters [Progress Report]
Environmental Factors Affecting Use of Ribotyping to Identify Fecal Contamination Sources in Estuaries [Progress Report]
Field Testing Targeted Sampling and Enterococcus faecalis to Identify Human Fecal Contamination in Three National Estuarine Research Reserves [Progress Report]