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玉米检疫性种传细菌免疫学及基因芯片检测方法的研究

标题: 玉米检疫性种传细菌免疫学及基因芯片检测方法的研究
英文标题: Reseach on Immunosorbant Assay and Gene-chip Methods for Seedborn Bacterias in Corn Seeds
作者: 吴晓巍
出版时间: 2010-01-01
所在大学: 河北农业大学
关键词: 玉米细菌性枯萎病菌,玉米内州萎蔫病菌,基因芯片,ELISA
英文关键词: Pantoea stwartii,Clavibacter. subsp.nebraskensis,Gene-chip,ELISA
论文级别: 硕士
学位: 学位论文
导师: 董金皋%田世民
专业: 植物病理学
提交时间: 2010
摘要: 玉米细菌性枯萎病(Bacterial Wilt of Corn or Stewart's Wilt, Pantoea stwartii)和玉米内州萎蔫病(Goss's bacterial wilt and blight of maize, Clavibacter. subsp.nebraskensis)是玉米生产上的两种重要病害,在北美州国家或地区有大面积发生的报道.我国尚未有两种病害发生的报道,已被列入我国进境植物检疫性有害生物名录中.因此,建立一套灵敏、高效的检验检疫方法显得尤为重要.两种病原菌的常用检测方法有分离培养、免疫学、PCR检测方法.目前还没有玉米内州萎蔫病菌单克隆抗体在免疫学检测方面应用的报道,.基因芯片检测方法是一种世界上最先进便携式生物化学分析器的核心技术,与上述三种检测方法相比,检测灵敏度高,且可实现高通量、并行性的检测目的.因此,本论文在实验室现有玉米内州萎蔫病菌单克隆抗体的基础上,建立了双单抗夹心ELISA检测方法,填补了目前国内外无该病菌单抗检测方法的空白,并为后期检测试剂盒及试纸条的研制奠定了基础.对两种玉米检疫性细菌及其相关病菌进行基因芯片检测研究,建立了高通量、并行性的基因芯片检测方法.结果如下:1对筛选到的玉米内州萎蔫病菌单克隆抗体进行亚类、效价等指标的测定,并对ELISA检测中抗原、抗体测试浓度进行了筛选.2建立了双单抗夹心ELISA检测方法,检测灵敏度可达1.0*108 CFU/L,并进行了玉米种子样品添加测试.实验结果表明,该检测方法重复性好且稳定性高.3设计筛选了针对两种病原细菌的特异性引物及探针,以及与玉米内州萎蔫病菌属同一亚种的番茄溃疡病菌(Clavibacter michiganensis subsp.michiganensis)、苜蓿细菌性萎蔫病菌(Clavibacter michiganensis subsp.insidiosus)的通用引物及相应的探针,建立了四种病原菌的多重PCR扩增方法,可以有效扩增出各菌株的目的基因片段.4制备了针对四种目标病原菌的基因芯片,并对各测试条件进行了筛选,确立了各阶段的最适反应条件.5建立了四种病原菌的基因芯片检测方法,该方法实现了多种病原菌的同时检测,其检测灵敏度高、特异性好,结果可直接通过肉眼观测,且在样品检测过程中受基质影响很小,检测效率高.本研究建立的双单抗夹心检测方法和基因芯片检测方法,在一定程度上均可满足对口岸进境玉米中细菌性枯萎病菌和内州萎蔫病菌的检测要求.相比较而言,基因芯片检测方法的灵敏度和检测效率比ELISA检测方法略高,但由于基因芯片检测成本交较高,在一定程度上,可应用ELISA检测方法进行初检,以降低检测成本,对阳性样品或可疑结果样品进行基因芯片复检,两种检测方法配合使用,实现准确、高效、经济的检测目的.
英文摘要: Bacterial Wilt of Corn or Stewart's Wilt (Pantoea stwartii), and Goss's bacterial wilt and blight of maize (Clavibacter michiganensis subsp. nebraskensis, Cmn ) are important disease classes in corn production. Currently they are taking places in most part of North America.Therefore these two bacterial diseases have already recoreded in been in quarantine test list for Chinese entry plant, despite of that they have not been still reported in China. Nowadays, it is very important to establish a sensitive and efficient testing method for the inspection and detection of the couple of corn diseases.There are three detection methods for these two bacterium which are separated culture, serological and PCR detection method.There are no reports of the Cmn monoclonal antibodies in serological detection. Compared with the polyclonal antibodies, monoclonal antibodies showed specificity, high purity and stability. Microarray method is one of the world's most advanced core technology of portable biochemical analyzer, Comparing with routine separated culture and PCR detection methord, Gene-chips expressed a high detection sensitivity, high throughput and high parallelism capacity, which can meet with a precise detection requirement. Therefore, in this article, on basis of Cmn monoclonal antibodies, we established a double-monoclonal-antibody sandwich ELISA method for detection of the Cmn, which filled up the blank of monoclonal-antibody detection methods in-around the world, and settled the basis for preparation of ELISA kit and test strip in the next step. By Gene-chip study on the two quarantine bacterium of maize and related bacteria, a Geng-chip detection method was developed, the method can realize simultaneous detection of multiple bacterium.The results were summarized as follows:1 Subclasses, activity, and specificity of the Cmn-McAb were test, and the optimum condition for antigen and antibody were screened.2 A noval DAS-ELISA method was established with its minimum detection limit of the bacteria was 1.0*108 CFU/L, and the bacteria in the seed suspension with high repeatability and stability had been detected.3 Primers and probes for Pantoea stwartii, Cmn, Clavibacter michiganensis subsp.michiganensis (Cmm) and Clavibacter michiganensis subsp.insidiosus (Cmi) were designed. And a specific and sensitive multiple PCR of the bacteria was developed.4 The visual gene chips were preparation and the best conditions for the detection were screened.5 A specific and sensitive visual Gene-chips detection methord was established. It can simultaneous of two or more individual bacteria. Little influences in matrix during the process of sample testing were observed.In this study, we developed two new detection methods of DAS-ELISA and Gene-chip for corn bacteria diseases. They can meet with part of the requirements for entry maize testing. The detection sensitivity and efficiency of gene chip are better than the ELISA method. Yet the ELISA method is relatively cheap. In order to reduce testing cost, ELISA method can be used firstly with the redetection for the positive samples arranging in pair with the two detection methods to achieve accurate, efficient and economic detection results.