Development of high sensitive monitoring method of toxic dinoflagellates by molecular probes

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Toxic thecate dinoflagellates of the genus Alexandrium, especially A.tamarense and A.catenella, produce potent neurotoxins such as gonyautoxin and saxitoxin responsible for paralytic shellfish poisoning (PSP) in costal weaters and cause serious problems in publich health and fishery. The morphological taxonomy of these microalgae, however, remains controversial because of the similarity and changeability of morphological features. Accordingly, a objective and precise method for identification is really reqiured. In this study I established the base of monitoring of these toxic species by flow cytometry using a monoclonal antibody and DNA probes. The results are as follows ;(1) Monocal antibody M8751-1 reacted with the cells of A.tamarense 5-10 times stronger than those of A.catenella. The labeled cells with the antibody could be efficiently identified and quantified by fluoresence microscope and flow cytometer. The antibody did not react with other harmful microalgae Gymnodinium catenatum, G.mikimotoi, Chattonella marina and Heterosigma akashiwo.(2) The antibody reacted with the cells of A.tamarense in natural field samples and the labelled cells were able to detect by flow cytometry. Any amplication of the antibody reactivity need to detect the field cells of A.catenella.(3) Fluorescent DNA probes complementary to the 3'end of ITS1 (internal transcribed spacer of rDNA) of A.tamarense (tam probe) and A.catenella (cat probe) reacted with each targeted species and did not react with other related species. These DNA probes hava high applicability as a general "tag" for identification of toxic Alexandrium species.