| 徐 匆，黄 皓，罗华建，杜见南，黄晓彦，陈 彦，梁卫驱，胡 珊.一步法 RT-LAMP-HNB 检测兰花建兰花叶病毒和齿舌兰环斑病毒方法的建立[J].广东农业科学,2022,49(5):95-101 |
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一步法 RT-LAMP-HNB 检测兰花建兰花叶病毒和齿舌兰环斑病毒方法的建立 |
| Establishment of RT-LAMP-HNB Method for Detection of Cymbidium mosaic Virus and Odontoglossum Ringspot Virus |
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| DOI：10.16768/j.issn.1004-874X.2022.05.011 |
| 中文关键词: 建兰花叶病毒 齿舌兰环斑病毒 反转录 - 环介导等温扩增（RT-LAMP） 羟基萘酚蓝（HNB） cp 基因 |
| 英文关键词: Cymbidium mosaic virus Odontoglossum ringspot virus RT-LAMP hydroxynaphthol blue(HNB) cp gene |
| 基金项目:东莞市社会科技发展重点项目（2020507101164） |
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| 中文摘要: |
| 【目的】全世界兰花病毒有 50 余种，建兰花叶病毒（Cymbidium mosaic virus，CymMV）和齿舌兰
环斑病毒（Odontoglossum ringspot virus, ORSV）是其中最常见的两种病毒。目前仍没有有效的措施能够控制病毒
病，因此建立针对病毒的高效监测、检测方法，对控制兰花病毒感染具有至关重要的意义。【方法】以兰花病
毒 CymMV 和 ORSV 为试验材料，采用反转录 - 环介导等温扩增方法（RT-LAMP）进行病毒 RNA 的扩增，从而
建立 CymMV 和 ORSV 的快速检测方法。分别根据两种病毒的 cp 基因序列保守区设计 RT-LAMP 引物，在一步
法 RT-LAMP 反应体系中加入羟基萘酚蓝（HNB，终浓度 200 µmol/L）作为扩增产物指示剂，不需开盖进行凝胶
电泳检测，便可直接根据体系颜色变化判读反应结果。【结果】利用该检测方法能够在 2 h 测出样品中是否存在
CymMV 和 ORSV 病毒 RNA，其灵敏度是 RT-PCR 的 10 倍。对 20 株田间样品进行一步法 RT-LAMP-HNB 检测，
根据田间检测结果显示，CymMV 检出率为 70%，ORSV 检出率为 55%，与 RT-PCR 结果保持一致，该法适用于
田间样品检测。【结论】建立的一步法 RT-LAMP-HNB 是一种灵敏、快速、特异、便捷的 CymMV 和 ORSV 检
测方法，仅需要简单控温设备如水浴锅便可进行核酸扩增，适用于在基层或不便利地区推广使用。 |
| 英文摘要: |
| 【Objective】There are more than 50 kinds of orchid viruses in the world, among which Cymbidium
mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the most common viruses. However, there are
no effective measures to control the virus disease. Therefore, the establishment of efficient detection methods for the
CymMV and ORSV is of great significance to control infected plants.【Method】CymMV and ORSV were used as
experimental materials, and the reverse transcription loop-mediated isothermal amplification method (RT-LAMP)
was used to amplify the viral RNA, so as to establish a rapid detection method of CymMV and ORSV. The RT-LAMP
primers were designed according to the conserved regions of the cp gene sequences of two viruses. The hydroxylnaphthol blue (HNB, the final concentration of 200 μmol/L）was added to the RT-LAMP reaction system as an indicator of the
amplification product. Without opening the tube for gel electrophoresis, the reaction results could be directly interpreted by
the color change of the system which could avoid product aerosol pollution.【Result】The detection method could detect
the presence of CymMV and ORSV RNA in the samples within 2 h, and its sensitivity was 10 times that of RT-PCR. In
order to test the detection effect of RT-LAMP-HNB in actual production, 20 Phalaenopsis samples were detected by onestep RT-LAMP-HNB. According to the results, the detection rates of CymMV and ORSV were 70% and 55%, which
were consistent with the results of RT-PCR, indicating that this method was suitable for the detection of field samples.
【Conclusion】The one-step RT-LAMP-HNB established in this study is a sensitive, rapid, specific and convenient method
for the detection of CymMV and ORSV. Nucleic acid amplification can be carried out only by simple temperature control
equipment such as water bath pot, suitable for promotion and use in grass-roots or inconvenient areas. |
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