以tuf基因为靶标的5种16SrⅠ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 16SrⅠ Group Based on Target tuf Gene

【目的】建立一种能够特异性检测和鉴定16SrⅠ组植原体的环介导恒温扩增技术,实现16SrⅠ组植原体的快速检测。【方法】以植原体tuf基因作为靶标,通过序列比对,设计多组具有特异性的LAMP引物组,筛选出一套适用于16SrⅠ组植原体的特异性检测体系。以16SrⅡ组、16SrⅤ组、16SrⅩⅨ组植原体样品按照此检测体系进行反应,来验证其特异性;同时将稀释后的感病组织样品同步进行PCR和LAMP检测,以确定其检测灵敏度。对来自不同省市的6份田间样品以及9份组培样品进行检测,以验证其检测的稳定性。【结果】16SrⅠ-LAMP在恒温63℃条件下40 min内完成扩增,能检测5种分别引起泡桐丛枝、苦楝丛枝、桑树萎缩、莴苣黄化和长春花绿变病的16SrⅠ组植原体,不能检测其他组植原体包括16SrⅡ组的花生丛枝、甘薯丛枝、臭矢菜丛枝、16SrⅤ组的枣疯病、红花槐丛枝、樱桃致死黄化和16SrⅩⅨ组的板栗黄化皱缩植原体。通过在反应液中加入钙黄绿素肉眼判断绿色为阳性结果,褐色为阴性结果,与用荧光定量设备进行判读扩增曲线的结果一致。相比于PCR检测,16SrⅠ-LAMP检测的灵敏度提高了8倍;16SrⅠ-LAMP能够快速检测出来自不同区域的田间泡桐发病样品、感病泡桐组培苗和嫁接传病脱毒苗。【结论】以tuf基因作为靶标,建立能同时检测5种16SrⅠ组植原体的环介导恒温扩增技术,具有高效、特异、操作简便、检测时间短及成本较低等优点,适合于基层和现场检测。

【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 16 Sr Ⅰgroup 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 atconstant 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 16 Sr ⅩⅨ （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 Pa WB 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 grassroots and field testing and for the rapid diagnosis of phytoplasma associated diseases.