逆转录环介导等温扩增技术检测齿兰环斑病毒

樊荣辉; 罗远华; 钟淮钦; 叶秀仙; 黄敏玲

doi:10.19303/j.issn.1008-0384.2019.07.011

逆转录环介导等温扩增技术检测齿兰环斑病毒

RT-LAMP Detection of Odontoglossum Ringspot Virus

摘要

摘要:
目的齿兰环斑病毒Odontoglossum ringspot virus（ORSV）是侵染兰花的主要病毒，严重影响其观赏价值，建立快速、灵敏的检测方法显得尤为重要。
方法本研究根据ORSV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（Reverse Transcription-Loop-mediated Iisothermal Amplification）检测方法。
结果结果显示该方法能特异扩增ORSV，与其他4种侵染兰花的病毒（建兰花叶病毒、菜豆黄花叶病毒、黄瓜花叶病毒和小苍兰花叶病毒）不发生反应；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。在产物中加入荧光染料SYBR GreenⅠ直接用肉眼观察即可判断样品是否感染ORSV，省去电泳分析的时间，并且增加了在田间的应用价值。
结论该方法具有特异性强、灵敏度高、操作简单、快速等特点。

Abstract:
Objective To establish a rapid and reliable detection method on Odontoglossum ringspot virus (ORSV), a major pathogen that seriously affects the ornamental value of orchids.
Method Primers were designed for the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay from the conserved region in the coat protein (CP) gene of ORSV available in GenBank, and its reaction conditions optimized.
Result Using the selected primer, RT-LAMP specifically identified ORSV but not Cymbidium mosaic virus (CyMV), Bean yellow mosaic virus (BYMV), Freesia mosaic virus (FreMV) or Cucumber mosaic virus (CMV). The sensitivity of the assay was 10 times higher than that of RT-PCR, while identical in positive rate on test of 20 specimens. Furthermore, the amplification products of the newly developed method could be visually inspected using SYBR Green I without gel electrophoresis.
Conclusion The RT-LAMP assay was considered a specific, sensitive, and rapid method for ORSV detection.

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