TECHNOLOGY TRANSFER: HOW TO PERFORM THE MICROPLATE ASSAY, A MOLECULAR DIAGNOSTIC TECHNIQUE

1. HOW TO PERFORM THE ASSAY PICTORIAL OVERVIEW: Step 1: Design Molecular Probes A B 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 5’-CTCATGGTGGCGGCTGG-3’ design probes to be species-specific and to work at a single temperature Step 2: Immobilize Probes LEARN TO PERFORM THE ASSAY: 5’- CTCATGGTGGCGGCTGG -3’ Watch the Video at: http://ciceet.unh.edu/project_extras/microplate_assay A web portal has been established by CICEET to provide written, pictorial, and step-by-step video instruction on how to perform this molecular method. The information on this site can be of value to science educators as well as to researchers wanting to employ the technique. You may also find this information at: http://www.aoml.noaa.gov/ocd/Kelly-II/index.html probe + = T-tailed probe to raise probe off plate surface TTTTTTTTTTT Brevis Probe: 2 bp specificity needed to distinguish K. brevis from K. mikimotoi poly-t’s TECHNOLOGY TRANSFER: HOW TO PERFORM THE MICROPLATE ASSAY, A MOLECULAR DIAGNOSTIC TECHNIQUE Kelly D. Goodwin NOAA Atlantic Oceanographic and Meteorological Laboratories BACKGROUND covalent chemistry Harmful Algae = and immobilize probe to wells finished microplate Step 3: Extract & Amplify DNA Sewage Contamination Impact Coastal Water A) filter water sample Water Quality Assays Need Improvement B) extract genomic DNA • Harmful Algae: • require extensive microscopic expertise • hard to distinguish closely related species • samples fragile & hard to preserve probe: target: ColinSitu ENTI Bdis C) PCR with biotin-labeled primers Escherichia coli Enterobacter aerogenes Klebsiella pneumonia Enterococcus faecalis Pseudomonas aeruginosa Bacteroides distasonis Bacteroides vulgatus PCR blank Prevotella ruminicola • Fecal Contamination: • labor intensive • take too long • measure indicators vs. pathogens Probes for a variety of fecal bacteria. Colinsitu for E. coli, ENTI for total coliforms, and Bdis for Bacteroides distasonis, an anaerobe associated with the human gut. DVDs of the instructional video are available free of charge. Step 4: Hybridize DNA Molecular-Based Assays: Blue +Stop Solution = Yellow REFERENCES Enzyme Substrate • Sensitive • Specific • Microscope & Culture Independent Kiesling,T., M. Diaz, A. Statzell-Tallman, and J.W. Fell (2002) Field identification of marine yeasts using DNA hybridization macroarrays. In: Marine Mycology:  The Organisms, Ecology and Applied Aspects. Hyde,K. (ed). Hong Kong: Fungal Diversity Press, 69-80. K.D. Goodwin, S.A. Cotton, G. Scorzetti, and J.W. Fell (2005) A DNA hybridization assay to identify toxic dinoflagellates in coastal waters: detection of Karenia Brevis in the Rookery Bay National Estuarine Research Reserve. Harmful Algae, 4:411-422 Streptavidin-POD Target DNA Biotin Probe Development Goals: • aid managers and decision makers • aid ecological research • provide early and accurate detection • fast, convenient, economical Microplate Well yellow color identifies presence of organism Example Microplates: OVERVIEW A DNA hybridization assay in microtiter format (the “microplate assay”) can be used to detect problem organisms in coastal water samples. Species-specific probes are immobilized onto the surface of 96-well plates. The technique yields rapid colorimetric results which can be accessed by visual inspection or quantified by a colorimetric plate reader. The assay can be adapted to a variety of targets, including bacteria and functional genes. The Cooperative Institute for Coastal and Estuarine Environmental Technology (CICEET) arranged for a market analysis to be performed on the technique. The market analysis recommended knowledge transfer targeting “do-it-yourself” end-users. The recommendation suggested that end-users would be capable of adapting the technology to suit their purposes if given adequately detailed information (e.g., more than can be obtained from a publication). A web portal and instructional DVD were established to facilitate knowledge transfer.