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

有毒甲藻高灵敏分子探针监测方法的建立

• 批准号: 07660244
• 负责人: SAKO Yoshihiko
• 金额: $ 1.28万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660244/
• 关键词: Toxic dinoflagellate; Alexandrium; Paralytic shellfish poisoning; Saxitoxin; Gonyautoxin; DNA probe; Molecular identification; Flow cytometry; モノクローナル抗体; リボソームRNA; 種間・種内識別

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.

亚历山大藻属（Alexandiums）甲藻，特别是塔玛亚历山大藻（A.tamarense）和链状亚历山大藻（A.catenella），能产生神经毒素，如膝尾藻毒素（gonyautoxin）和石房蛤毒素（saxitoxin），引起沿岸鱼类麻痹性贝毒（PSP），严重危害鱼类健康和渔业生产。然而，由于这些微藻形态特征的相似性和易变性，它们的形态分类仍存在争议。因此，需要一种客观、准确的识别方法。在这项研究中，我建立了使用单克隆抗体和DNA探针的流式细胞术监测这些有毒物质的基础。结果表明：（1）单抗M8751-1与塔玛曲霉细胞的反应强度是链状曲霉的5-10倍。荧光显微镜和流式细胞仪可对抗体标记的细胞进行有效的鉴定和定量。该抗体与其它有害微藻链状裸藻、米氏裸藻、海洋查顿藻和赤潮异弯藻均不发生反应。(2)该抗体与自然野外样本中的塔玛曲霉细胞发生反应，标记的细胞能够通过流式细胞术进行检测。抗体反应性的任何扩增都需要检测链状放线菌的野细胞。(3)与塔玛曲霉（tam探针）和链状曲霉（cat探针）的ITS 1（rDNA的内部转录间隔区）3 '末端互补的荧光DNA探针与每个靶物种反应，而不与其他相关物种反应。这些DNA探针具有很高的适用性，可作为鉴定有毒亚历山大藻的通用“标签”。

项目成果

Adachi M. et al.: "Population analysis of dinoflagellate Alexandrium species using sequences of the 5.8S ribosomal DNA and internal transcribed spacer regions." J. phycol.31(in press). (1996)

Adachi M. 等人：“使用 5.8S 核糖体 DNA 和内部转录间隔区序列对甲藻亚历山大藻进行种群分析。”

K.Nakanishi et al.: "Detection of the red tide-causing plankton Alexandrium affine by a piezoelectric immunosensor using a novel method of immobilizing antibodies" Analytical Letters. 29(8). 1247-1258 (1996)

K.Nakanishi 等人：“使用固定抗体的新方法通过压电免疫传感器检测引起赤潮的浮游生物亚历山大藻”分析快报。

Sako Y. et al.: "Detection of the toxic dinoflagellates Alexandriumspecies by flow cytometry using a monoclonal antibody" Toxic Phytoplankton. 7(in press). (1996)

Sako Y. 等人：“使用单克隆抗体通过流式细胞术检测有毒的甲藻亚历山大藻”有毒浮游植物。

M.Adachi et al.: "Identification of the toxic dinoflagellates Alexandrium catenella and A.tamarense (Dinophyceae) using DNA probes and whole-cell hybridization" J.Phycol.32. 1049-1052 (1996)

M.Adachi 等人：“利用 DNA 探针和全细胞杂交鉴定有毒甲藻链状亚历山大藻和 A.tamarense（甲藻纲）”J.Phycol.32。

M.Adachi et al.: "Analysis of Alexandrium (Dinophyceae) species using sequences of the 5.8S ribosomal DNA and internal spacer regions" J.Phycol.32. 424-432 (1996)

M.Adachi 等人：“使用 5.8S 核糖体 DNA 和内部间隔区序列分析亚历山大藻（甲藻科）物种”J.Phycol.32。