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

【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。

【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 one-step 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.