【作者】 徐霞；

【导师】 廖晓兰；

【作者基本信息】 湖南农业大学 ， 生物安全与检疫， 2014， 硕士

【摘要】 根据寄主植物的不同将密执安棒状杆菌分为五个亚种：番茄细菌性溃疡病菌(Cmm),侵染引起番茄溃疡病；马铃薯环腐病菌(Cms),侵染引起马铃薯环腐病；苜蓿细菌性萎蔫病菌(Cmi),侵染引起苜蓿细菌性萎蔫病；玉米萎蔫病菌(Cmn),侵染引起玉米内州萎蔫病；小麦细菌性花叶病菌(Cmt),侵染引起小麦细菌性花叶病。这些病害常造成巨大的经济损失,其中番茄细菌性溃疡病菌、苜蓿细菌性萎蔫病菌、玉米内州萎蔫病菌为我国进境检疫性有害生物。目前,针对密执安棒状杆菌植物病菌还没有快速可行的检测方法,严重影响了口岸检疫的效率。因此,针对该种植物病菌建立快速、灵敏的检测方法对防止病害的发生、传播与流行具有重要的意义。本文针对密执安棒状杆菌(Clavibacter michiganensis)种内五个病菌建立了交叉引物恒温扩增(Cross Priming Amplification, CPA)检测体系,并通过结合可视性胶体金检测试纸条,建立了一种具有简单、快捷、灵敏特异、不需要昂贵仪器等优点的新型检测方法,可用于细菌性植物病害的快速筛查和检测。针对密执安棒状杆菌(Clavibacter michiganensis)种内五个植物病菌(Cmm、Cms、 Cmi、Cmn、Cmt)的ITS基因序列设计了三条引物及两条探针,应用上述方法对密执安棒状杆菌植物病菌进行检测,并同时应用普通PCR法、实时荧光定量PCR法进行检测。结果显示应用CPA检测方法能够从纯培养菌悬液中准确检测到目标病原菌,五个病菌的灵敏度分别为Cmm:1.32×104cfu/mL; Cms:1.48×104cfu/mL; Cmi:4.75×103cfu/mL; Cmn:2.35×104cfu/mL; Cmt:1.78×103cfu/mL。灵敏度与实时荧光定量PCR相当,远远优于普通PCR检测技术。在带菌种子实体检测研究中,分别以人工模拟的番茄种子和番茄种子样品浸泡液中提取的DNA为模板,利用本实验建立的CPA检测方法成功检测出番茄溃疡病菌且灵敏度达到5.62××105cfu/mL。本研究建立的密执安棒状杆菌植物病菌交叉引物恒温扩增检测方法,只需要简单的加热和离心仪器,可以直接目视实验结果,具有准确、灵敏、快捷的优点,能有效弥补传统PCR技术、实时荧光PCR技术在仪器设备不完善的基层科研机构、口岸检疫无法应用的不足,但是本实验建立的交叉引物恒温扩增方法还不能区分密执安棒状杆菌的5个亚种,在引物设计上还需进一步完善。更多还原

【Abstract】 All known subspecies of Clavibacter michiganensis (Cm) are subdivided into five subspecies:Clavibacter michiganensis subsp. michiganensis (Cmm) is the casual agent of bacterial wilt and canker of tomato; Clavibacter michiganensis subsp. sepedonicus (Cms) give rise to potato ring rot; Clavibacter michiganensis subsp. insidious (Cmi) causes wilting and stunting in alfalfa; Wilt and blight of maize is induced by Clavibacter michiganensis subsp. nebraskensis (Cmn) and Clavibacter michiganensis subsp. tessellarius (Cmt) causes leaf freckles and pots in wheat. Among the five bacteria, Clavibacter michiganensis subsp. michiganensis (Cmm)、Clavibacter michiganensis subsp. insidious (Cmi) and Clavibacter michiganensis subsp. nebraskensis are quarantine pests for our country. Therefore, it is very essential to develop rapid and sensitive diagnostic methods to detect these bacteria and limit the effects of diseases.In this paper, foused on the five pathogens of Clavibacter michiganensis, a simple, rapid, sensitive and specific detection system has been established based on CPA (Cross Priming Amplification) combined with gold-labeled DNA strip sensor. This diagnostic method does not need any expensive instruments and can be used to screen for bacterial plant disese.Three primers and two probes were developed based on the ITS sequences of Clavibacter michiganensis subspecies. This protocol was used to specially detect Clavibacter michiganensis plant pathogens and the results were compared and identified by routine PCR and real-time PCR respecitively. The assay showed that the CPA method can specifically detect the object strains from pure bacterial culture, the sensitivity of the five pathogens was as follow, Cmm:1.32×104cfu/mL; Cms:1.48×104cfu/mL; Cmi:4.75×103cfu/mL; Cmn:2.35×104cfu/mL; Cmt:1.78×103cfu/mL. The sentivity was almost the same as real-time PCR and higher than routine PCR.In the detection experiment of tomato seeds, as few as5.62×105cfu/mL can be successfully detected with sample DNA extraction from practically and artificially infected tomato seeds. The research established the detection method for Clavibacter michiganensis plant pathogens based on cross priming amplification (CPA) techonology. In the entire detection procedure, from sample processing to visual readout, all the instruments needed are heater and centrifuge. The new method created in this paper made the whole detection procedure more accurate, sensitive and rapid. It can effectively make up for the disadvanges of routine PCR and real-time PCR at the grassroots level. However, the method based on CPA in this research cannot distinguish the five subspecies; it has significant popularizing value and application prospect.更多还原