High-throughput Detection For Three Major Bacterial Pathogens On Tomato

As the bacterial pathogens of Clavibacter michiganense subsp. michiganse (CMM),Pseudomonas syringae pv.tomato (PST) and Ralstonia solanacearum (RS) could causeserious diseases of tomato, the padlock probe for CMM, PST and RS was designedrespectively based on the species-specific DNA fragment in their complete genome.While the universal primers which came from Xist gene of mice were designed accordingto the linking sequence in the middle of padlock probe. By optimizing the reactionsystem, the rolling circle amplification detection system was proposed and comparedwith conventional PCR on specificity and sensitivity. Then these three bacterialpathogens were taken as the research objects. The reverse dot blot hybridizationtechnique which was based on the padlock probe and rolling circle amplification wasused to establish a high-throughput detection system, and then the sensitivity andspecificity of the system were tested accordingly.The results showed that based on the padlock probe design principle and specificsequence of CMM, PST and RS, the three padlock probes were designed accordingly.The further tests showed that they had high specificity and sensitivity. The detectionsystem on the optimization could detect the above three pathogens specifically only,while it did not work for other plant pathogens. The sensitivity of rolling circleamplification detection system was500fg/μL, which was10-fold higher than that ofconventional PCR. While the rolling circle amplification system labeled with digoxigeninworking together with reverse dot blot hybridization technology could detect CMM, PSTand RS in one reaction, providing a new method for high throughput detection of thethree pathogens initially.