As the coastal human population increases in the United States, there will likely be increasing environmental and socioeconomic pressures on our coastal and estuarine environments. Monitoring the condition of all our nation's coastal and estuarine ecosystems over the long term is more than any one program can accomplish on its own. Therefore, it is crucial that monitoring programs at all levels (local, state, and federal) cooperate in the collection, sharing, and use of environmental data. This volume is the proceedings of the Coastal Monitoring Through Partnerships symposium that was held in Pensacola, Florida in April of 2001, and was organized by the U.S. Environmental Protection Agency's (EPA's) Environmental Monitoring and Assessment Program (EMAP), and the Council of State Governments (CSG). It contains papers that describe various multi-disciplinary coastal and estuarine environmental monitoring programs, designed and implemented by using regional and national partnerships with federal and state agencies, academia, Native American tribes, and nongovernmental organizations. In addition, it includes papers on modeling and data management; monitoring and assessment of benthic communities; development of biological indicators and interlaboratory sediment comparisons; microbiological modeling and indicators; and monitoring and assessment of phytoplankton and submerged aquatic vegetation. There are many components involved in determining the overall impacts of anthropogenic stressors on coastal and estuarine waters. It will take strong partnerships like those described in this volume to ensure that we have healthy and sustainable coastal and estuarine environments, now and in the future.
Two methods for the detection of important human pathogens, Cryptosporidium parvum and Helicobacter pylori, were investigated: a fiber optic biosensor, and real time PCR. The mechanism for specific detection in both methods is recognition of specific DNA sequences in the target organisms. The biosensor that was used, the Analyte 2000, was originally developed for the detection of chemicals. It utilizes a fiber optic wave guide that propagates an evanescent light wave of very specific wavelength. The light excites fluorescent molecules bound to the waveguide, but not in the bulk solution, which theoretically enhances signal while reducing background interference. Attempts to develop this system for the detection of DNA were not successful due to poor detection of the target molecules. An assay analogous to a sandwich immunoassay was designed for use on the Analyte 2000. Specific oligonucleotide probes were designed to bind to the waveguides via biotin-streptavidin interaction, and were used to capture the target DNA. Pure target DNA representing unique genes in the organisms were synthesized by PCR. Detection of captured DNA was then attempted using an oligonucleotide detection probe designed to bind to the target. Two detection systems were employed: an indirect signal amplification system based on biotin-tyramide deposition, or direct detection of fluorescent signal from Cy-5 molecules. In all experiments performed there was very little difference between the signal generated with or without the target molecules. Many experiments were conducted to attempt to identify reasons for the poor signal. Signal was only of any significance when target amplicons were internally labeled with Cy-5 by PCR. Real time PCR as a method to detect the pathogens was also investigated. Though the PCR technique itself is very rapid, DNA extraction and purification requires preparation time. Filtration of up to one liter of well water, followed by concentration and "cleaning" Helicobacter pylori cells by immunomagnetic separation, was used to detect H. pylori seeded in a water source. Following cell lysis, the extracted DNA could be used directly in conventional PCR targeting the 16S rRNA gene to detect less than 265 cells per liter of water. DNA purification was not required for this level of detection. Initial studies to amplify lysed cells by real time PCR indicated that an incorrect product was made. When purified DNA was used for real time PCR, the correct product was produced from DNA representing as few as 100 cells. This publication can be purchased and downloaded via Pay Per View on Water Intelligence Online - click on the Pay Per View icon below
As the coastal human population increases in the United States, there will likely be increasing environmental and socioeconomic pressures on our coastal and estuarine environments. Monitoring the condition of all our nation's coastal and estuarine ecosystems over the long term is more than any one program can accomplish on its own. Therefore, it is crucial that monitoring programs at all levels (local, state, and federal) cooperate in the collection, sharing, and use of environmental data.
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