Abstract: 【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 based on padlock probe for DNA concentration was about 50 fg•μL-1, while the sensitivity of conventional PCR method was about 5 pg•μL-1, the former was 100 times higher than the latter. To detect tomato samples for CMM, 5 samples were tested positive from 45 samples, among which 2 samples were from Japan (Jap1214, Jap1102), 2 samples from Yongtai (Yongt1001, Yongt1002) and 1 sample from Minqing (Minq1001). 【Conclusion】The assay of real-time PCR based on padlock probe was specific and sensitive, and the detection method was used to test the entry tomato seeds collected, CMM could be detected directly from tomato seeds. This method would be suitable for CMM rapid detection of tomato seeds imported, and would be an effective tool for detecting CMM in entry-exit phytosanitary.

Key words: Clavibacter michiganensis subsp. michiganensis , padlock probe , real-time PCR , hyperbranched rolling circle amplification , rapid detection , tomato seed