中文摘要：

【目的】从口岸进境番茄种子中检测番茄溃疡病菌（Clavibacter michiganensis subsp. michiganensis，CMM），一直以来都受检测周期的限制，快速、特异地从种子中检测该病原细菌需要新的方法。研究在锁式探针扩增方式上，选择连接酶依赖的PCR扩增方式，并结合实时荧光PCR技术，旨在建立番茄溃疡病菌基于锁式探针技术的实时荧光PCR快速检测方法，为口岸番茄种子快速检疫提供技术支持。【方法】根据番茄溃疡病菌的一段特异基因Pat-1序列，设计锁式探针的T1和T2臂，使之与CMM的特异性片段碱基序列互补，再按照锁式探针的设计原则设计锁式探针和扩增引物以及荧光探针。试验时，先将锁式探针与CMM以及参照菌株的DNA模板在DNA连接酶作用下分别进行环化连接，再用核酸外切酶Ⅰ和核酸外切酶Ⅲ消化未环化的线性锁式探针，最后以环化的锁式探针为模板，在扩增引物的作用下进行实时荧光PCR试验。建立CMM基于锁式探针技术的实时荧光PCR检测方法，分别比较该方法的特异性和灵敏度与常规PCR方法的差异，并用该方法对收集的来自日本、韩国和中国台湾的番茄种子以及国内采集的共45份样品进行检测。【结果】基于锁式探针技术的实时荧光PCR检测方法能够从供试的菌株中特异性地检出CMM，在供试的10种菌株中，只有靶标细菌能被特异性地检测到阳性，空白对照和其他参试菌株均没有荧光信号的增加，检测为阴性。比较该检测方法与常规PCR方法，其特异性和常规PCR方法一致。该检测方法检测灵敏度高，DNA最低浓度检测为50 fg·μL-1，而常规PCR方法检测DNA最低浓度为5 pg·μL-1，灵敏度高于常规PCR两个数量级。对收集的样品进行检测，结果显示，45份样品中共有5份样品CMM检测结果为阳性，分别是日本的番茄种子样品2份（编号Jap1214、Jap1102），永泰采集的?

英文摘要：

【Objective】Detection of tomato canker （Clavibacter michiganensis subsp. michiganensis, CMM） from imported tomato seeds has been restricted to the detection time, therefore, a new approach is needed to detect CMM rapidly and specifically from tomato seeds. In this study, combined with real-time PCR technology, the ligase-dependent PCR amplification was carried out in amplification way of padlock probe. The objective of this study is to establish the method of real-time PCR based on the padlock probe for detecting CMM, and to provide technical supports for entry-exit phytosanitary. 【Method】 T1 and T2 arms of the padlock probe, which were complementary to the specific nucleotide sequence of CMM, were designed based on the sequence of the unique hypothetic protein gene Pat-1 in complete genome of CMM. The universal primers and TaqMAN fluorescent probes were also designed according to the design principles of padlock probes. During the test, firstly, padlock probe was connected with the DNA of CMM and other reference strains under the action of the DNA enzyme, respectively, then, the connected product padlock probe was digested with exonucleaseⅠand Ⅲ to remove uncyclized linear padlock probe, finally, real-time PCR amplification was tested with the Cyclized padlock probes as a DNA template. The detection system for CMM by using real-time PCR based on padlock probe was established, the specificity and sensitivity of this method were compared with the conventional PCR method, respectively, and a total of 45 tomato samples were detected using this method, which were collected from Japan, Korea and China’s Taiwan, as well as the main land of China. 【Result】 CMM could be detected specifically from the 10 tested strains with real-time PCR based on padlock probe method, only the target bacteria could be specifically detected positive, the blank control and other strains were detected negative. The specificity was consistent with the conventional PCR method. The detection sensitivity of real-time PCR bas