Abstract: [Objective]The purpose of this study is to develop an isothermalmethod known as loop-mediated isothermal amplification (LAMP) for detection of 16SrⅠ group phytoplasmas. The method would make it possible to achieve simple and rapid detection of this pathogen.[Method]In present study, several sets of LAMP primers were designed using the tuf gene as the target, and then the most appropriate set was selected to develop LAMPmethod for detection of 16SrⅠ group phytoplasmas. The specificity of LAMP was tested by using 3 other groups of phytoplasma (16SrⅡ, 16Sr V, 16Sr XIX) which are closely related to 16SrⅠ group. The sensitivities between LAMP and PCR for detecting phytoplasma were compared by using two-fold serially diluted DNA extracted from phytoplasma-infected paulownia as templates. The LAMP method was used to detect the paulownia witches'-broom phytoplasmas from 6 provinces in China and 9 kinds of tissue culture seedlings.[Result]The 16SrⅠ-LAMP was efficiently amplified with the target tuf gene sequences in 40 min at constant temperature of 63℃, by which five 16SrⅠ group phytoplasmas causing paulownia witches'-broom, chinaberry witches'-broom, mulberry dwarf, lettuce yellows and periwinkle phyllody diseases were detected, but no phytoplasmas from 16SrⅡ (peanut witches'-broom, sweet potato witches'-broom, cleome witches'-broom), 16Sr V (jujube witches'-broom, cherry lethal yellows, Bischofia polycarpa witches'-broom, Robinia hispida witches'-broom), and 16SrXIX (chestnut yellows crinkle) as well as healthy plant control were detected. The result of LAMP were observed by the color changes of reaction solution added to calcein, the reaction solution was green for positive samples and orange for negative ones, which were in agreement with the result by detecting amplification curve through the fluorescent quantitation device. Compared with conventional PCR amplification, the detection sensitivity of 16SrⅠ-LAMP was 8-fold higher. The PaWB samples collected from different regions and different graft-inoculted tissue culture seedlings could be detected correctly with corresponding LAMP assay.[Conclusion]This is the first report of application of the LAMP assay technique for the simple,efficient,and specific detection of 16SrⅠ group phytoplasmas targeting on phytoplasma tuf gene. suitable for grass-roots and field testing and for the rapid diagnosis of phytoplasma associated diseases.

Key words: phytoplasma, loop mediated isothermal amplification(LAMP), tuf gene, rapid detection