Detection Of Didymella Bryoniae Using The Loop-Mediated Isothermal Amplification And Padlock Probe Based Technology

The study of plant pathogen detection and identification techniques is the focus of inspection and quarantine work of national border agencies. Since it can prevent the invasion of alien species and protect the economic security of agro forestry production activities and ecological security, these techniques have significant contributions. So far the existing traditional detection technologies and molecular detection techniques, such as, conventional PCR technique is unable to satisfy the above requirements, hence, the search of more stable and specific detection targets, and the establishment of new detection technology system, have become increasingly important.This research by using two molecular detection method:Loop-mediated isothermal amplification (LAMP) technology and Padlock probe (combined with Macroaary) technology to detect the pathogenic fungi(Didymella bryoniae) of Gummy Stem Blight and establish their efficient detection system. The target site in this study of the LAMP primers and the Padlock probe is one of housekeeping genes: RNA polymerase Ⅱ (RPB2) gene, and we took experimental verification for the sensitivity and specificity of the primers and probe. The result of experiment shows that the primers and probe can achieve the sensitive goal which is specific pathogenic fungi can be detected by specificity, and the sensitivity of detection can reach to200fg, which is the minimum concentration threshold for the DNA reaction system. The sensitivity of detection is100times to ordinary PCR detection method and fully meet the requirements of inspection and quarantine work.The loop-mediated isothermal amplification technique used in this study, only takes about one hour to do the detection. This method has high sensitivity, and the reaction does not involve the degeneration and annealing of templates sequences. Therefore, there is no need of the thermal cycler in the process. Instead of that, constant temperature water-bath is used to complete the experiment and the final reaction results are obviously. Meanwhile, Padlock probe technique also has the characteristics of short time consuming, high sensitivity and easy operation. If it is combined with Macroaary, the experiment results become visible by reflecting a color reaction to ensure the result to be specificity. All these results below suggest that both of the two rapid molecular detection technologies are efficient, reliable.convenient, and provide a new choice for the port inspection and quarantine work in future.