【作者】 王念武；

【导师】 胡方平；

【作者基本信息】 福建农林大学 ， 植物病理学， 2013， 博士

【摘要】 番茄是我国重要的经济蔬菜之一，番茄上病害很多，达40余种，其中包括3种重要的细菌性病菌，即，番茄细菌性斑点病菌（Pseudomonas syringaepv.tomato）、番茄细菌性溃疡病菌（Clavibacter michiganensis subsp.michiganensis）和番茄青枯病菌（Ralstonia solanacearum），其中Cmm和Pst是欧洲和地中海植物保护组织（EPPO）检疫性有害生物（EPPO/CABI，1997），也是我国检疫性细菌，在我国局部发生，对当地的番茄种植产生很大的影响，造成了严重的经济损失。近年来，随着国外优良番茄种子的引进，番茄细菌病害传入我国的风险加大，口岸针对番茄细菌病原的检疫力度和难度加大，很需要提供针对进境种子等繁殖材料的多病原高通量检测技术。本研究选取Cmm、Pst和Rs13种重要番茄病原细菌为研究对象，根据Dreier等、Fanelli等和Lee等分别获得的Cmm，Pst和Rs1的一段特异性片段设计锁式探针的T1和T2臂，再根据锁式探针的设计原则，设计通用引物、杂交探针和TaqMAN荧光探针。实验时，分别进行了3种病原细菌的超分支滚环扩增检测研究，基于锁式探针的反向斑点杂交检测技术研究，基于锁式探针的实时荧光PCR检测技术研究和基于锁式探针的DNA芯片检测技术研究，并在这些研究的基础上，运用这些检测技术对福建口岸截获的番茄种子进行高通量检测。1、建立了Cmm、Pst和Rs1三种细菌的超分支滚环扩增检测技术，并对其特异性和灵敏度进行了测试。结果显示：（1）超分支滚环扩增体系优化时，当使用Taq DNA连接酶，采用热循环连接法，可以得到很好的扩增效果；锁式探针终浓度为8pmol/L时，连接效果最好；超分支滚环扩增温度为62℃，扩增效果最佳；采用10U的核酸外切酶Ⅰ和5U核酸外切酶Ⅲ来消化未连接的线性探针，能有效消除线性探针的影响。（2）三种细菌的超分支滚环扩增的特异性很强，在供试的菌种中，只有靶标细菌能被特异性的检测到，其他的菌株检测为阴性，实验中，用常规PCR的特异性引物进性验证，与超分支滚环扩增的特异性基本一致。（3）三种细菌的超分支滚环扩增的灵敏度高，最低检测DNA浓度为500fg/μL，高于常规PCR的检测灵敏度，比常规PCR灵敏度高出1个数量级。2、建立了三种细菌的基于锁式探针技术的反向斑点杂交高通检测技术，实现了同一反应体系内同步高通量检测。结果显示：（1）在供试的菌株中，该技术能在同一反应体系内一次性检测多种病原物。（2）基于锁式探针技术的反向斑点杂交特异性强，实验中，用基于锁式探针的连接酶依赖性PCR扩增，代替滚环扩增进行反向斑点杂交，在供试的菌种中，只有靶标细菌能被特异性的检测到，其他的菌株检测为阴性。（3）基于锁式探针技术的反向斑点杂交灵敏度高，最低检测DNA浓度为5pg/μL，与常规PCR检测灵敏度相当。3、建立基于锁式探针技术的实时荧光PCR方法。结果显示：（1）该方法能从供试的菌株中分别检测出番茄上三种病原物；（2）基于锁式探针技术的实时荧光PCR技术特异性强，在供试的菌种中，只有靶标细菌能被特异性的检测到，其他的菌株检测为阴性；（3）基于锁式探针技术的实时荧光PCR方法灵敏度高，最低检测DNA浓度为50fg/μL，比常规PCR检测灵敏度高出两个数量级。4、建立基于锁式探针技术的DNA芯片检测方法。结果显示：（1）该方法能从供试的10菌株中分别检测出番茄上三种病原物；（2）基于锁式探针技术的DNA芯片检测方法特异性强，在供试的菌种中，只有靶标细菌能被特异性的检测到，其他的菌株检测均为为阴性；（3）基于锁式探针技术的DNA芯片检测方法灵敏度高，最低检测DNA浓度为500fg/μL，比常规PCR检测灵敏度高出1个数量级；（4）混合锁式探针与混合DNA的DNA芯片检测，能特异地检出靶标菌，适合口岸番茄种子的多病原高通量检测。5、用这些检测技术对番茄种子进行应用检测，对模拟的带菌番茄种子进行超分支滚环扩增，并对口岸对收集到的45份样品分别用基于锁式探针的实时荧光PCR技术、反向斑点杂交技术和DNA芯片技术分别进行检测。结果显示：（1）用Cmm使健康番茄种子模拟带菌，通过超分支滚环扩增的方法，可以检测出模拟菌Cmm，由此可推知，带菌番茄种子是可以被检测出的，该模拟方法具有实际意义；（2）用基于锁式探针技术的实时荧光PCR技术对供试的45份样品进行Cmm检测，有5份样品为阳性，其它的样品为阴性；（3）基于锁式探针技术的反向斑点杂交检测45份样品，共检测出Cmm阳性样品5份，Pst阳性样品5份，Rs1阳性样品8份，其它的样品结果均为阴性；（4）基于锁式探针技术的DNA芯片检测45份样品，共检测出Cmm阳性样品5份，Pst阳性样品5份，Rs1阳性样品8份，其它的样品结果均为阴性；（5）基于锁式探针的实时荧光PCR技术、反向斑点杂交技术和DNA芯片技术均能检测样品，且检测的结果基本一致，互相验证了方法的可靠性。更多还原

【Abstract】 Tomato is one of the important economic vegetable in Chinese vegetable.Thediseases in tomato are more than40kinds,including three important bacterial species,namely, tomato bacterial spot disease (Pseudomonas syringae pv.tomato), tomatobacterial canker (Clavibacter michiganensis subsp. michiganensis) and tomatobacterial wilt (Ralstonia solanacearum race1). Cmm and Pst, which have occurredlocally in our country and caused serious economic losses, are quarantine pests ofEuropean and Mediterranean Plant Protection Orgnization (EPPO), and are alsochinese quarantine bacteria. In recent years, with the introduction of foreign excellenttomato seeds, the risk of tomato diseases introducing into our country has beenincreased. It is necessary for the entry of seeds and other propagating material toapply high throughput multi-pathogen detection technology.In this assay, Cmm，Pst and Rs1were selected as the research bacteria, accordingto the specific gene fragments of Cmm, Pst and Rs1obtained by Dreier, Fanelli, andLee, respectively, the T1and T2arms of their padlock probes were designed. And, theuniversal primers, hybridization probes and TaqMAN fluorescent probes were alsodesigned, according to the design principles of padlock probes. Cmm，Pst and Rs1were tested with four methods, which were the method of hyperbranched rolling circleamplification (HRCA), reverse dot-blot hybridization based on padlock probe,real-time PCR based on padlock probe,and DNA chip based on padlock probe. On thebasis of these studies, tomato samples intercepted in Fujian ports were tested forhigh-throughput detection.1, The detection technology of HCRA for Cmm, Pst and Rs1were established,and its specificity and sensitivity were also tested. The results showed that:(1) inHCRA optimization system, when using Taq DNA ligase, and using thermal cyclingconnection method, a good amplification effect could be got；the connection effectwas the best when final concentration of padlock probe was about8pmol/L；theamplification effect was the best at62℃；when using a10U exonuclease Ⅰ and 5Uexonuclease Ⅲ to digest the uncyclized padlock, the impact of linear padlockprobe could be removed effectively.(2) the specificity of HRCA was very high, onlythe target bacteria that could be detected specifically, while, the other strains weretested negative, the specificity of this method was compared with the conventionalPCR method, the result showed that the specificities of two methods were consistent.(3) the sensitivity of HRCA was very high, the minimum concentration of DNAdetected was500fg/μL, which was10-fold higher than that of conventional PCR.2, The high-throughpu detection technology for Cmm, Pst and Rs1wasestablished, using method of reverse dot-blot hybridization based on padlock probe.The results showed that:(1) multiple pathogens could be detected by this method inthe same reaction system.(2) the method of reverse dot-blot hybridization based onpadlock probe had high specificity, only the specific target bacteria that could bedetected from the test strains, the other strains tested negative.(3) the detectionsensitivity of reverse dot-blot hybridization based on padlock probe was about5pg/μL, which was coincident with that of conventional PCR.3, The detection technology of real-time PCR based on padlock probe for Cmm,Pst and Rs1was established, respectively.The results showed that:(1) Cmm, Pst andRs1could be detected from the tested strains by the method of real-time PCR basedon padlock probe, respectively.(2) the method of real-time PCR based on padlockprobe had high specificity, only the specific target bacteria that could be detected fromthe test strains, the other strains tested negative.(3) the detection sensitivity of thismethod was very high, the minimum concentration of detected DNA was50fg/μL,which was100-fold higher than that of conventional PCR.4, The detection method of DNA chip based on padlock probe was established.The results showed that:(1) Cmm, Pst and Rs1could be detected from the10testedstrains by this method, respectively.(2) the method of DNA chip based on padlockprobe had high specificity, only the specific target bacteria that could be detected fromthe test strains, the other strains tested negative.(3) the detection sensitivity of thismethod was high, the minimum concentration of DNA was about500fg/μL, whichwas10-fold higher than that of conventional PCR.(4) the target bacteria could bedetected from the tested bacteria by this method when padlock probes were linked between mixed lock probes and mixed DNA, the assay of DNA chip based on padlockprobe was specific and sensitive, and was suitable for high-throughput detection ofpathogens, it would be an effective tool for detecting Cmm, Pst and Rs1in entry-exitphytosanitary.5, In application detection for tomato samples, the simulated tomato seedscontaminated by Cmm were tested by the method of HRCA, and45collected sampleswere tested by the method of reverse dot-blot hybridization based on padlock probe,real-time PCR based on padlock probe, and DNA chip based on padlock probe,respectively. The results showed that:(1) Cmm could be detected by the method ofHRCA from the simulated tomato seeds contaminated by Cmm, which could beinferred that the contaminated tomato seeds could be detected and the simulationdetection method had the practical significance.(2) in the samples detection for CMMwith the method of real-time PCR based on padlock probe,5samples tested positivefrom45samples, which were2samples from Japan(Jap1214, Jap1102),2samplesfrom Yongtai(Yongt1001, Yongt1002) and1sample from Minqing (Minq1001), theother strains were tested negative.(3) in the detection for Cmm,Pst and Rs1with themethod of reverse dot-blot hybridization based on padlock probe,5Cmm samples,5Pst samples and8Rs1samples tested positive from the45samples, the other samplestested negative.(4) in the detection for Cmm,Pst and Rs1with the method of DNAchip based on padlock probe,5Cmm samples,5Pst samples and8Rs1samplestested positive from the45samples, the other samples tested negative.(5) tomatosamples could be detected with three methods, which were the real-time PCR, reversedot-blot hybridization and DNA chip based on padlock probe, and the test results bythe three methods were identical, testing and verifying mutually the detectionreliability.更多还原