【作者】 夏明星；

【导师】 马青；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2005， 硕士

【摘要】 番茄细菌性溃疡病(Clavibacter michiganensis subsp. michiganensis,Cmm)是严重危害番茄的细菌性病害,主要靠带菌种子进行远距离传播,被许多国家列为检疫病害。在口岸检疫及种子种苗健康检测中,传统的分离培养、生理生化鉴定、噬菌体检测、免疫荧光染色及寄主致病性试验等是研究者一直采用的方法。这些检验方法存在很多局限性,不适合口岸检疫快速验放的要求。因此,快速、灵敏、特异性强的PCR 技术近年来被广泛应用于植物病原菌的检测。但常规PCR 技术需很多后续处理,也容易造成实验室及PCR 产物的污染。TaqMan 实时荧光PCR 技术是在普通PCR 基础上加入一条TaqMan探针,该探针能在PCR 的每个循环与靶序列进行特异性杂交,有效提高反应特异性和灵敏度,更好地避免假阴性结果,使PCR 技术检测痕量样品的能力进一步加强。本研究依据番茄细菌性溃疡病菌ITS序列的多态性设计TaqMan 探针及特异性引物,分别对该病菌进行常规PCR 及实时荧光PCR 检测。特异性引物及TaqMan探针能检测出所有供试的Cmm 菌,其它对照菌均无电泳条带也未收集到荧光信号,显示出该引物-探针组合有很强的特异性。通过对不同浓度菌悬液的常规PCR 和实时荧光PCR 扩增,发现实时荧光PCR 检测灵敏度比常规PCR 高约100 倍,能很好地避免假阴性结果。以接种但未显示症状的番茄苗叶片提取核酸作为模板,常规PCR 方法能在接种后第6 天检测到该菌,而实时荧光PCR 则可以在接种后第4 天就检测到该菌的存在。通过对人工模拟带菌种子的测试发现,番茄种子的核酸作为模板对PCR 扩增不存在抑制作用或抑制作用微弱,完全可以通过实时荧光PCR 技术获得准确的试验结果。将带菌种子浸泡液及带菌种子的核酸作为模板进行PCR 扩增,实时荧光PCR 的相对灵敏度明显高于常规PCR,而且经过比较得出,应用种子核酸作为模板能到达更好的检测效果。本研究可直接应用植物总DNA作为试验材料,无需进行病原菌的分离培养及性状观察。由于采用光电传导系统和计算机操作系统能够很直观地反映出整个PCR 过程各个反应阶段的信息,而且不需要进行PCR 产物的后续处理,可以提高检测效率,减少污染。本研究在国内首次建立了对Cmm 的TaqMan实时荧光PCR 检测体系,并获得很好的检测结果。该方法快速、灵敏、特异、安全,能及早提供准确的病害信息,为农业生产部门、检疫部门及种子种苗相关领域提供有力的技术支持。更多还原

【Abstract】 Clavibacter michiganensis subsp. michiganensis is the most destructive bacterial pathogen of tomato. The infected seeds are the major way to spread the pathogen distantly. EPPO has listed Cmm as an A2 quarantine pest. The traditional methods such as: isolation and culture, physiological and biochemical identification, phage specificity, serological test and host reaction-were applied to detect and identify the bacterium. These methods are time-consuming, low efficiency,low accuracy and sensitivity, which can not meet the custom-quarantine expectations. PCR was applied to detect the phytopathogen widely in recently years, which is more rapid and efficient. However, the gel electrophoresis is needed to determine the sizes of amplified product after normal PCR and the contamination, safety and time-consuming problems have been noted as significant issues. So a safer, more accurate, sensitive and rapid PCR method are required for phytopathogen detection. The series of primers and Taqman probe were designed based on the region of ITS to detect Cmm with normal PCR and Real-Time fluorescent PCR (RTF PCR) in this experiment. The specific primers and probes were found that they could detect all the Cmm isolates collected from different sources. The protocol was approximately 100 times more sensitive than normal PCR and the amplified products were confirmed by specific fluorescent probe used during amplification. Strong fluorescent signal could be collected in the reaction when the DNA were directly extracted from the inoculated seed and seedling which had no symptom. Furthermore, the pathogen isolation is not required and contamination is controlled because the whole detection process is finished in the contained tubes. This method can provide a specific, sensitive, rapid detection of Cmm for quarantine department and the seed industry.更多还原