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| 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 |
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| Abstract: |
| 【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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