the microplate assay provided identification of several toxic dinoflagellates, including karenia...
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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
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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
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