Microplate Assay Development for Detecting Microbial Contaminants



 

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08/2003
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Project Title: Assay and Sensor Development to Identify, Detect, and Quantify Microbial Contaminants
Research Locations: Rookery Bay, Naples FL
Last Update: 10-28-03


Challenge

Decisions to close shellfish beds and beaches due to microbial contaminants in coastal waters have economic repercussions that can be felt on local, regional, and national levels. In order to make these important determinations, managers need accurate, quick, and cost-effective methods for pinpointing the types and levels of contaminants. Traditional culture-based microbial methods are slow, labor intensive, and do not differentiate between human and animal sources of waste. This project is developing a microplate assay that quickly and accurately identifies species of toxic plankton and fecal-indicating bacteria.



Science


Methods: The Microplate Assay
Researchers tested two groups of organisms: toxic dinoflagellates that cause red tide, and bacteria that indicate the presence of sewage. They first identified the molecular sequences needed to detect the problem organisms. From there, they created "molecular probes" which they attached to the wells of microplates. DNA from a sample was then placed in the well. If there was a match, a chemical reaction caused a yellow color to indicate the presence of that organism in the water sample.


Preliminary results

  • The red tide assay successfully detected Karenia brevisin water samples collected from the Rookery Bay NERR. The assay was sensitive enough to detect "very low" and "present" amounts of this toxic dinoflagellate.
  • The sewage-indicating assay utilized a variety of probes. Probes were developed to detect 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. to be carried out simultaneously, each one being completed in less than a minute.



Application

Convenient, rapid relay of information
Pre-existing methods require up to 3 days for analysis and relay of information to resource managers. The microplate assay can give managers information in a matter of 2-3 hours. Using this method would make the process quicker, less expensive, and more informative. It would allow for easy storage of samples and would aid in differentiating between human and non-human fecal pollution sources.

In the Future: An even faster technique
Results of this project have been so successful researchers are now developing a new and even faster technique for identifying microbial contaminants which would allow for hundreds of tests to be carried out simultaneously, each one being completed in less than a minute.



Essentials

Project Coordinator

Dr. Jack Fell (305) 361-4603
jfell@rsmas.miami.edu
University of Miami

Dr. Kelly Goodwin (305) 361-4384
kelly.goodwin@noaa.gov
National Atlantic Oceanic and Meteorologic Laboratories

Start -End Date:

01/09/2002 - 01/09/2004

NERR Reserve(s):

Rookery Bay, Naples FL

For more information: Dolores Jalbert Leonard
Phone: (603) 862-3685
Email: dolores.leonard@unh.edu