樊荣辉, 黄敏玲*, 钟淮钦, 叶秀仙, 罗远华.环介导等温扩增技术检测小苍兰花叶病毒[J].植物保护,2019,45(2):153-156. |
环介导等温扩增技术检测小苍兰花叶病毒 |
Establishment of a RT-LAMP assay for detection of Freesia mosaic virus |
投稿时间:2018-04-11 修订日期:2018-05-23 |
DOI:10.16688/j.zwbh.2018164 |
中文关键词: 小苍兰花叶病毒 RT-LAMP 检测 |
英文关键词:Freesia mosaic virus RT-LAMP detection |
基金项目:省属公益类科研院所基本科研专项(2018R1026-6);福建省农业科学院科技创新团队项目(STIT2017-2-9) |
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中文摘要: |
小苍兰花叶病毒Freesia mosaic virus (FreMV)是侵染兰花的主要病毒, 严重影响其观赏价值。本研究根据FreMV的外壳蛋白基因序列设计了一组特异性引物, 经过一系列条件优化, 建立了该病毒的RT-LAMP检测方法。结果显示设计引物能特异扩增FreMV, 与其他4种病毒(菜豆黄花叶病毒Bean yellow mosaic virus、黄瓜花叶病毒Cucumber mosaic virus、建兰花叶病毒Cymbidium mosaic virus和齿兰环斑病毒Odontoglossum ringspot virus)不发生反应; 该方法灵敏度为RT-PCR的10倍。田间检测20份样品中, RT-LAMP和RT-PCR检测结果一致, 检出率为60%。在产物中加入荧光染料SYBR GreenⅠ直接用肉眼观察就可判断样品是否感染FreMV, 可省去电泳分析的时间。该方法具有特异性强、灵敏度高、操作简单、快速等特点。 |
英文摘要: |
Freesia mosaic virus (FreMV) is one of the main viruses infecting orchids, seriously affecting the ornamental value. In order to establish a reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay, the primers were designed based on the conserved region of the coat protein(CP) gene of FreMV available in GenBank, and the reaction conditions were optimized. FreMV was specifically identified by RT-LAMP by using these primers, but Bean yellow mosaic virus, Cucumber mosaic virus, Cymbidium mosaic virus and Odontoglossum ringspot virus were not amplified. The sensitivity of the RT-LAMP assay was 10-fold higher than that of RT-PCR.The positive rate of 20 samples by using the RT-LAMP was 60%, which was consistent to that of RT-PCR, validating the accuracy of the RT-LAMP assay. In addition, PCR products could be visualized by using SYBR Green Ⅰ, and gel electrophoresis was not a necessity. Hence, this RT-LAMP assay is a specific, sensitive and rapid method for detecting FreMV. |
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