Guangdong nongye kexue (May 2022)

Establishment of RT-LAMP-HNB Method for Detection of Cymbidium mosaic Virus and Odontoglossum Ringspot Virus

  • Cong XU,
  • Hao HUANG,
  • Huajian LUO,
  • Jiannan DU,
  • Xiaoyan HUANG,
  • Yan CHEN,
  • Weiqu LIANG,
  • Shan HU

DOI
https://doi.org/10.16768/j.issn.1004-874X.2022.05.011
Journal volume & issue
Vol. 49, no. 5
pp. 95 – 101

Abstract

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【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.

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