The microplate assay provided identification of several toxic dinoflagellates, including Karenia brevis, the organism responsible for red tide in the Rookery Bay NERR (Fig. 1). The assay was evaluated with 110 environmental samples and was consistent with species-specific PCR, sequencing, and microscopic observation. Target sensitivity for this study was a negative result consistent with a report of “not present” provided by a regional monitoring program. With HPLC purified probes, this objective was met in every case tested. Assay sensitivity allowed detection of K. brevis when it was “present” in the water (<1000 cells/L), as defined by the Florida Marine Research Institute (FMRI). Simultaneous and species-specific detection of multiple targets was demonstrated with K. brevis and Amphidinium carterae and was achieved with a single hybridization condition and one set of PCR primers. Immobilized “Karenia” probe detected both K. brevis and Karenia mikimotoi (Fig. 2). The “Brevis” probe distinguished between these closely related species (Fig. 3).

RESULTS

The microplate assay: Immobilized DNA Probes to Rapidly Detect Toxic Dinoflagellates Kelly D. Goodwin*, Sara A. Cotton+, Gloria Scorzetti**, Traci Kiesling **, and Jack W. Fell**

*NOAA Atlantic Oceanographic and Meteorological Laboratories, +Coop. Inst. Marine & Atmos. Studies & **Marine Biology and Fisheries Rosenstiel School of Marine and Atmospheric Science, University of Miami

A DNA hybridization assay in microtiter plate format

was adapted to detect toxic dinoflagellates and fecal

bacteria in coastal waters. The assay provided species-

specific identification and simultaneous detection of

multiple targets. The assay detected K. brevis in coastal

waters collected from the Rookery Bay National

Estuarine Research Reserve (NERR). Results were

verified by species-specific PCR and sequence analysis.

The presence/absence of K. brevis was consistent with

microscopic observation. The assay yielded quick

colorimetric results, employed a single hybridization

temperature, and conserved the amount of genomic

DNA utilized by using one set of PCR primers. The

microplate assay provides a useful tool to quickly screen

large sample sets for multiple target organisms.

ABSTRACT

REFERENCES1based on Regnault et al. Res. Microbiol. 2000, V151, pp. 521-533, 2based on Loge et al. Water Env. Res. 1999, 71:76-83; 3based on Franks et al. Appl. Environ. Microbiol. 1998, 64: 3336-3345; 4Kiesling, T.L., Wilkinson, E., Rabalais, J., Ortner, P.B., McCabe, M.M. and Fell, J.W., Mar. Biotechnol. 2002, 4:30-39.

Detection of Toxic Dinoflagellates

METHODS

5’-CTCATGGTGGCGGCTGG-3’

design probes to be species-specific and to work at a single temperature

Step 1: Design Molecular Probes

T-tailed probe to raise probeoff plate surface

=

5’- CTCATGGTGGCGGCTGG -3’probe + TTTTTTTTTTT

poly-t’s

=

finished microplate

covalent chemistry

immobilize probe to wells

Step 2: Produce Microplates

Step 4: Hybridize DNA

yellow color identifies presence of organism

Streptavidin-POD

Enzyme Substrate

Biotin Target DNA

Probe

Microplate Well

Blue + Stop Solution = Yellow

Step 3: Extract & Amplify DNA A) filter water sample

C) PCR with biotin-labeled

universal primers

B) extract genomic DNA

Fig. 2 Karenia Probe: Detects K. brevis and K. mikimotoi

Karenia brevis, CCMP 718 Karenia brevis, TX-sp3 Karenia brevis, NOAA-1Karenia mikimotoi, NOAA-2 negative PCR control

blank

A B C D

Fig. 3. Brevis Probe: 2 bp specificity needed to distinguish

K. brevis from K. mikimotoi

Karenia brevis, CCMP 718, 100%Karenia brevis, CCMP 718, 50%Karenia brevis, CCMP 718, 25% Karenia brevis, TX-sp3 Karenia brevis, NOAA-1 Karenia mikimotoi, NOAA-2 negative PCR control

blank

A B

Fig. 1. Sampling sites in the Rookery Bay NERR

1

2 3

4 5 67 8

1) Henderson Creek 2) Marco Pass 3) 951 boat ramp 4) Caxambas Pass 5) Goodland 6) Blackwater River 7) Faka Union Bay 8) Fakahatchee Bay

The microplate assay allows rapid identification of toxic dinoflagellates and fecal bacteria without the microscopic expertise and culturing normally required. The assay can detect multiple species simultaneously and distinguish between closely related species. The technique does not require expensive instrumentation and has several advantages over species-specific PCR or cloning and sequencing of total extracted DNA. The assay gives 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. The technique conserves the amount of genomic DNA utilized, which can be critical to certain applications4. The microplate assay offers the sensitivity and specificity of molecular analysis in a convenient, adaptable, and relatively inexpensive format.

CONCLUSTIONS

BACKGROUND

Harmful Algae:• require extensive

microscopic expertise• hard to distinguish

closely related species• samples fragile & hard

to preserveFecal Contamination:

•labor intensive •take too long •measure indicators

vs. pathogens

Water Quality Assays Need Improvement

Molecular-Based Assays:SensitiveSpecificMicroscope & Culture Independent

aid managers and decision makersaid ecological researchprovide early and accurate detectiondistinguish between human and animal wastefast, convenient, economical

Development Goals:

Harmful Algae

Sewage Contamination

Impact Coastal Water

and