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A detection method based on reverse transcription loop-mediated isothermal amplification for a genetically heterogeneous plantago asiatica mosaic virus

  • Viral and Viroid Diseases
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Abstract

A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect plantago asiatica mosaic virus (PlAMV), one of the most damaging lily-infecting viruses and a member of the genus Potexvirus in the family Alphaflexiviridae. A set of six primers was designed based on the central core region of the coat protein gene of the Li1 isolate of PlAMV, which detected the isolate most efficiently at 65 °C. The RT-LAMP assay specifically detected several PlAMV isolates with a high level of genetic and biological variation, but not potato virus X (another virus species in the same Potexvirus genus). The sensitivity of the RT-LAMP was tenfold higher than that of conventional RT-PCR. Moreover, with a simple method using a toothpick, PlAMV was directly detected from infected lily leaves using the RT-LAMP assay without RNA extraction. This simple and highly sensitive method can be used for rapid surveys for PlAMV.

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Acknowledgments

We thank Mr. T. Moriyama for providing lily plants. This work was supported in part by Grants-in-Aid for Young Scientists (B) (26850231) from the Japan Society for the Promotion of Science.

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Authors and Affiliations

  1. Laboratory of Plant Pathology, Tokyo University of Agriculture and Technology (TUAT), 3-5-8 Saiwaicho, Fuchu, Tokyo, 183-8509, Japan

    Ken Komatsu, Tsutomu Arie & Tohru Teraoka

  2. Laboratory of Plant Pathology, Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113-8657, Japan

    Kensaku Maejima, Naoko Fujita, Osamu Netsu, Tatsuya Tomomitsu & Shigetou Namba

Authors
  1. Ken Komatsu
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  2. Kensaku Maejima
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  3. Naoko Fujita
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  4. Osamu Netsu
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  5. Tatsuya Tomomitsu
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  6. Tsutomu Arie
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  7. Tohru Teraoka
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  8. Shigetou Namba
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Corresponding author

Correspondence to Shigetou Namba.

Electronic supplementary material

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10327_2015_599_MOESM1_ESM.pptx

Effect of healthy lily sap on RT-LAMP reaction. RT-LAMP amplification curves using total RNA (1 pg or 100 fg) isolated from a Li1-infected plant of Nicotiana benthamiana as the template are shown. In the sample marked “+ healthy lily sap”, a toothpick which had pricked healthy lily leaf (Fig. 5, healthy lily2) was dipped into the LAMP reaction mixture. RT-LAMP reaction was run for 50 min at 65 °C. (PPTX 49 kb)

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Komatsu, K., Maejima, K., Fujita, N. et al. A detection method based on reverse transcription loop-mediated isothermal amplification for a genetically heterogeneous plantago asiatica mosaic virus. J Gen Plant Pathol 81, 297–303 (2015). https://doi.org/10.1007/s10327-015-0599-6

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  • DOI: https://doi.org/10.1007/s10327-015-0599-6

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