Study on DNA probe specific for toxic dinoflagellate Alexandrium

产毒素甲藻属于链状亚历山大藻和塔玛亚历山大藻，能产生强烈的神经毒素，可引起近岸水域的麻痹性贝类中毒(PSP)，并造成严重的经济和公共卫生问题。然而，这些细菌的形态分类仍然存在争议，因为它们的特征非常相似和多变，而且不同菌株的毒素含量也不同。因此，迫切需要一种简单、容易和客观的鉴定方法。在这项研究中，我试图通过单克隆抗体来鉴定这两个物种，并开发针对每个物种的DNA探针。主要研究结果如下：(1)从塔纳部湾和大船渡湾分离的包囊中分离出萌发细胞，建立了卡氏弧菌和塔玛弧菌在克隆和无菌条件下的规模化培养。(2)用高效液相-荧光法分析了所有菌株的PSP曲霉毒素，然后对不同毒素含量的典型菌株进行了…分析。本实验首次建立了毒性亚历山大藻中高分子量全细胞DNA的纯化方法。(4)在有毒亚历山大藻中，首次建立了高分子量全细胞DNA的纯化方法。用17SrDNA的3‘端和24SrDNA的5’端的两对引物进行扩增，两种菌株的扩增产物均为610pb。DNA片段对应于ITS(内间隔区)1、5.8S和ITS2区。AluI和RsaI的酶切分析表明，这两个物种具有不同的限制性片段长度多态性。DNA片段鉴定结果与免疫学分析结果一致。从这些结果和两个物种ITS区较低的序列相似性来看，针对该区域的DNA探针绝对是鉴定有毒甲藻亚历山大藻的有效工具。较少

Toxigenic dinoflagellates belonging to Alexandrium catenella and A. tamarense produce potent neurotoxins responsible for paralytic shellfish poisoning (PSP) in coastal waters and cause serious economic and public health problems. However, the morphological taxonomy of these organisms remains controversial because of the very similar and changeable features and also toxin content is different among strains. Accordingly, a simple, easy and objective method for identification is a pressing need. In this study I attempted to identify both species by means of monoclonal antibodies, and to develop DNA probe specific for each species. The results are as follows;(1) Large scale cultures of A. catenella and A. tamarense with clonal and axenic conditions were established by the isolation of germinated cells from the cysts isolated from Tanabe Bay and Ofunato Bay.(2) PSP troxins of all strains were analyzed by HPLC-fluorometric analysis and then the typical strains showing different toxin content … More and composition were selected for following studies.(3) Monoclonal antibodies specific to toxic Alexandrium, which were prepared by us, could immunologically discriminate and identify inter- and intraspecies of these microalgae.(4) In toxic Alexandrium, the purification method of whole cell DNA with high molecular weight (>50kbp) was established for the first time. Amplification with two primers positioned 3'end of 17SrDNA and 5'end of 24SrDNA yielded a PCR product of 610pb in all strains of both species. The DNA fragments corresponded to the regions of ITS(internal spacer)1, 5.8S and ITS2. Digestion analysis with AluI and RsaI showed the different restriction fragment length polymorphism in both species. The identification results by the DNA fragments correspond to that of immunological analysis. From these results and also low degree of sequence similarity in the ITS regions of both species, DNA probe specific for this region is definitely an effective tool for the identification of toxic dinoflagellate Alexandrium species. Less