林业科学2017，Vol.53Issue(8)：54-63,10.DOI:10.11707/j.1001-7488.20170807
以tuf基因为靶标的5种16 SrⅠ组植原体环介导恒温扩增技术
Loop-Mediated Isothermal Amplification Assay for Detection of Five Phytoplasmas Belonging to 16SrⅠGroup Based on Target tuf Gene
摘要
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 16SrⅠgroup phytoplasmas. The specificity of LAMP was tested by using 3 other groups of phytoplasma (16SrⅡ,16SrⅤ,16SrⅩⅨ) 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),16SrⅤ (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.关键词
植原体/环介导恒温扩增/tuf基因/快速检测Key words
phytoplasma/loop mediated isothermal amplification(LAMP)/tuf gene/rapid detection分类
农业科技引用本文复制引用
王圣洁,王胜坤,林彩丽,于少帅,汪来发,朴春根,郭民伟,田国忠..以tuf基因为靶标的5种16 SrⅠ组植原体环介导恒温扩增技术[J].林业科学,2017,53(8):54-63,10.基金项目
"十二五"农村领域国家高技术研究发展科技计划(863)课题(2012AA101501) （863）
林业微生物资源子平台运行与服务项目(NIMR2016-7). （NIMR2016-7）
