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Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene

  • Bacterial and Phytoplasma Diseases
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Abstract

Phytoplasmas are plant pathogenic bacteria that infect more than 700 plant species. Because phytoplasma-resistant cultivars are not available for the vast majority of crops, the most common practice to prevent phytoplasma diseases is to remove infected plants. Therefore, developing a rapid, accurate diagnostic method to detect a phytoplasma infection is important. Here, we developed a phytoplasma detection assay based on loop-mediated isothermal amplification (LAMP) by targeting the groEL gene and 16S rDNA. We designed 19 primer sets for the LAMP assay and evaluated their amplification efficiency, sensitivity, and spectra to select the most suitable primer sets to detect Candidatus Phytoplasma asteris. As a result, DNA was efficiently amplified by one of the primer sets targeting the groEL gene, and LAMP assay sensitivity with this primer set was 10-fold higher than that of the polymerase chain reaction. Moreover, the groEL gene was successfully amplified from several strains of Ca. Phytoplasma asteris by this primer set, indicating that the groEL gene can be used as a LAMP assay target gene for a broad range of phytoplasma strains. Additionally, a simple DNA extraction method that omits the homogenizing and phenol extraction steps was combined with the LAMP assay to develop a simple, rapid, and convenient diagnostic method for detecting phytoplasma.

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Acknowledgements

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (category "A" of Scientific Research Grant 21248004), by the Funding Program for Next Generation World-Leading Researchers (project: GS005), and by the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN).

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Authors and Affiliations

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

    Kyoko Sugawara, Misako Himeno, Takuya Keima, Yugo Kitazawa, Kensaku Maejima, Kenro Oshima & Shigetou Namba

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  1. Kyoko Sugawara
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  2. Misako Himeno
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  3. Takuya Keima
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  4. Yugo Kitazawa
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  5. Kensaku Maejima
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  6. Kenro Oshima
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  7. Shigetou Namba
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Correspondence to Shigetou Namba.

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Sugawara, K., Himeno, M., Keima, T. et al. Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene. J Gen Plant Pathol 78, 389–397 (2012). https://doi.org/10.1007/s10327-012-0403-9

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  • DOI: https://doi.org/10.1007/s10327-012-0403-9

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