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

有毒的亚历山大属鞭毛藻，尤其是塔玛氏鞭毛藻和链鞭毛藻，会产生强效的神经毒素，如gonyautoxin和saxitoxin，导致沿海水域的麻痹性贝类中毒（PSP），并对公共卫生和渔业造成严重问题。然而，由于形态特征的相似性和可变性，这些微藻的形态分类仍然存在争议。因此，确实需要一种客观和精确的识别方法。在本研究中，我建立了利用单克隆抗体和DNA探针的流式细胞术监测这些有毒物种的基础。研究结果如下：(1)单抗M8751-1与A.tamarense细胞的反应强度是A.catenella细胞的5-10倍。用荧光显微镜和流式细胞仪对标记的细胞进行了高效的鉴定和定量。该抗体与其他有害微藻无反应性，如裸子藻、水藻、滨Chattonella和赤岛异角藻均无反应。(2)抗体与天然野样品中的tamarense细胞反应，标记的细胞可通过流式细胞术检测。(3)与A.tamarense （tam探针）和A.catenella （cat探针）的ITS1 （rDNA的内部转录间隔区）3'端互补的荧光DNA探针与每个靶种都发生反应，而与其他亲缘种不发生反应。这些DNA探针作为鉴定有毒亚历山大菌的通用“标签”具有很高的适用性。

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.