逆转录环介导等温扩增技术快速检测大豆花叶病毒  

Rapid Detection of Soybean Mosaic Virus by Reverse-Transcription Loop-Mediated Isothermal Amplification

作　　者：张雯娜[1] 李晋玉[1] 田金艳[1] 卫其巍[1] 智海剑[2] 陶小荣[1] 

机构地区：[1]南京农业大学植物保护学院,江苏南京210095 [2]南京农业大学大豆研究所/国家大豆改良中心,作物遗传与种质创新国家重点实验室,江苏南京210095

出　　处：《大豆科学》2014年第3期422-428,共7页Soybean Science

基　　金：国家高技术研究发展计划“863计划”(2012AA101501);公益性行业(农业)科研专项(201303028);教育部高校博士点基金(20130097110004);江苏省基础研究计划(自然科学基金)(BK2012769)

摘　　要：大豆花叶病毒（Soybean mosaic virus，SMV）是一种在世界很多国家广泛分布并极具破坏性的植物病毒，导致大豆严重减产和种质衰退，每年都会给很多国家带来巨大的农业经济损失，为此，建立了一种逆转录环介导等温扩增（ Reverse-transcription loop-mediated isothermal amplification, RT-LAMP）全新的SMV检测技术，利用该技术可以快速、准确与有效地检测大豆植株和种子中携带的SMV。根据GenBank数据库中大豆花叶病毒的外壳蛋白基因序列，设计并筛选到了SMV RT—LAMP特异性引物组，应用该引物组优化了RT—LAMP反应参数，灵敏度实验结果表明，RT-LAMP检测SMV比普通RT—PCR灵敏度高10～100倍。结合RNA粗提法，建立了一步法RT—LAMP快速、灵敏地检测到植物叶片中的SMV。加入荧光染料SYBR Green Ⅰ则可在反应管中直接观察检测结果，实现了检测的可视化。该RT-LAMP技术还可灵敏地检测到大豆种子中携带的SMV。RT-LAMP检测方法第一次应用于SMV的检测，具有灵敏度高、速度快和特异性强的优点，可应用于实验室和田间大豆样品SMV的快速检测。Soybean mosaic virus（SMV） is a widely distributed and highly destructive plant virus worldwide. Infection by SMV usually results in severe yield losses and seed quality reduction. SMV infected disease has caused a huge economic losses in many countries. There fore, reverse-transcription loop-mediated isothermal amplification（ RT-LAMP）, a method to achieve rapid, sensitive and accurate detection of SMV in soybean leaves and seeds was established. Using SMV coat protein gene sequences from GenBank database,the RT-LAMP primers sets were designed and the best primers set was selected. Then they were used for the optimization of the RT-LAMP parameters. The sensitivity of the RT-LAMP was 10-100 times higher than the conventional RT-PCR. When crude RNA was integrated in this assay, the developed one-step RT-LAMP could rapidly and sensitively detect SMV in soybean leaves. The reaction product could be directly visualized in tube by adding the fluorescent dye SYBR Ⅰ. The developed RT-LAMP could also successfully detect SMV in soybean seeds. This is the first report of the application of the RT-LAMP assay to detect SMV in soybean leaves and seeds. The rapid, sensitive and specific RT-LAMP assay developed in this study can be applied widely in laboratories and the field detection of SMV.

关 键 词：大豆花叶病毒 快速检测 RT—LAMP 大豆 种子带毒检测 

分 类 号：S435.651[农业科学—农业昆虫与害虫防治]