Molecular based in-situ, remote sensor for detecting microbial contaminants

Principal Investigators: Kelly Goodwin, Peter Ortner, Jack Fell (University of Miami)
Collaborating scientist(s): Sara Cotton

Objective: Develop an in-situ measurement system based on molecular indicators in order to relay the abundance of microbial contaminants in near real-time.

Rationale: The ecological balance of coastal waters is threatened by a variety of microbial contaminants including blooms of harmful algae, Vibrio bacteria, and pathogens derived from sewage waste. Decisions to close fisheries and beaches in order to protect human health are typically based on traditional assays, but improved assays are needed. Remote sensing has been viewed as an improved means to monitor coastal water quality and to understand basic ecological processes. However, the utility of remote sensing has been constrained by lack of species-specific data, which has continued to be labor-intensive to obtain. The goal of this study is to capitalize on advances in molecular biology to develop biotechnology that will improve water quality monitoring.

The frequency and occurrence of harmful algal blooms have increased. Consequences of such blooms include mortality of wild and farmed fish and shellfish, illness and death of humans and animals, and alteration of marine habitats. Blooms of non-toxic algae can also cause harm by causing habitat alteration, the displacement of indigenous species, oxygen depletion, and alteration of biogeochemical cycles. Virtually every coastal area in the United States is threatened by this problem, sometimes over large geographic areas and by more than one algal species. In Florida, Synechococcus is the dominant genus in the cyanobacterial blooms plaguing central Florida Bay. Karenia brevis is the responsible agent for recurrent red tides on the southwest Florida shelf. Improved monitoring is essential to understanding the factors that control bloom initiation and dynamics. Early and accurate warnings of contamination could protect human health by limiting the ingestion of contaminated fish and shellfish.

Decisions to close fisheries and beaches are based on water quality assays, but traditional microbiological assays suffer from serious drawbacks. Weakness include assays that are labor and supply intensive, samples that must be processed within 6-8 hr, results that are not available for over 24 hrs after the samples are processed, and assays that fail to differentiate between human and animal-derived wastes. Fecal-indicating bacteria also have a limited ability to accurately model the behavior of human pathogens in seawater. Cell count approaches used to monitor bloom forming algae and cyanobacteria also suffer practical drawbacks. Improved assays are needed to better protect human health and economic interests.

Methods: Initially, we will work with the toxic dinoflagellate Karenia brevis. K. brevis is an attractive organism with which to demonstrate proof-of-concept for the proposed technology because it is unicellular, unarmored (naked), and comparatively fragile. It is therefore amenable to reproducible, remotely controlled nucleic acid extraction. Species-specific probes will be designed based on sequence of the large subunit of the rDNA.

The proposed in situ sensor would use molecular indicators based on ampliprobe technology (AccuDx, patent pending). The ampliprobe system consists of an immobilized, unlabeled capture probe and a fluorescent-labeled signal probe that is added into solution during hybridization. The signal probes are unique in that they contain 100’s of fluorescent labels, greatly reducing the detection limit for the target. The design and function of the ampliprobe system will be optimized using a 96-well plate assay, producing an ancillary technology in the process.

This project will capitalize on a regional monitoring effort administered by Dr. Ortner (see http:// www.aoml.noaa.gov/ocd/sferpm/). The monitoring program includes a network of fixed platforms and buoys equipped with a suite of environmental sensors and remote telemetric data transmission. Field sites for this project will include Rookery Bay and NW Florida Bay. The molecular-based sensor would be integrated into the existing sensor network. The species-specific data generated by molecular-based sensors, in conjunction with synoptic measurement of relevant environmental variables, could significantly contribute to our understanding of the factors regulating bloom initiation. With adaptation (modification of probes and fluorescent dyes), the methodology should be equally applicable to other organisms and other littoral contexts, e.g., coastal upwelling, river plume stimulated plankton dynamics, other harmful algal bloom organisms, bacterial pathogens, etc.

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Last updated 09/21/2001 by the OCD Webmaster.