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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RTLAMP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), a plant virus that is widespread and devastating in many countries of the world, has led to severe soybean yield loss and germplasm decline, causing enormous agricultural economic losses to many countries every year Therefore, a new SMV detection technique based on Reverse-transcription Loop-mediated isothermal amplification (RT-LAMP) has been established, which can be used to detect soybean plants rapidly and accurately SMV carried in seeds. The SMV RT-LAMP specific primer set was designed and screened based on the coat protein gene sequence of soybean mosaic virus in the GenBank database. The RT-LAMP reaction parameters were optimized by using the primer set. Sensitivity experiments showed that RT-LAMP detected SMV ratio Ordinary RT-PCR high sensitivity 10 to 100 times. Combined with RNA crude extraction, a one-step RT-LAMP was established to detect SMV in plant leaves rapidly and sensitively. By adding the fluorescent dye SYBR Green I, the test results can be directly observed in the reaction tube to achieve the visualization of the test. The RTLAMP technology also allows the sensitive detection of SMV carried in soybean seeds. The first application of the RT-LAMP assay to SMV detection is the rapid, sensitive and rapid detection of SMV in laboratory and field soybean samples.