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Development and evaluation of a rapid, specific, and sensitive loop-mediated isothermal amplification assay to detect Tenacibaculum sp. strain pbs-1 associated with black-spot shell disease in Akoya pearl oysters

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Abstract

Black-spot shell disease decreases pearl quality and threatens pearl oyster survival. Establishment of a rapid, specific, and sensitive assay to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease is of commercial importance. We developed a rapid, specific, and highly sensitive loop-mediated isothermal amplification (LAMP) assay to detect Tenacibaculum sp. Pbs-1 in Akoya pearl oysters Pinctada fucata. A set of five specific primers (two inner, two outer, and a loop) were designed based on the 16S–23S internal spacer region of strain Pbs-1. The optimum reaction temperature was 63 °C, and concentrations of the inner and loop primers were 1.4 and 1.0 µM, respectively. The LAMP product can be detected using agarose gel electrophoresis, and the color change in the reaction tube can be detected visually (by the naked eye) following the addition of malachite green. Our assay proved to be specific for strain Pbs-1, with no cross-reactivity with five other species of Tenacibaculum. The detection limit of the LAMP assay at 35 min is 50 pg, and at 60 min it is 5 fg. We evaluated the LAMP assay using diseased and healthy pearl oysters. The results demonstrate the suitability and simplicity of this test for rapid field diagnosis of strain Pbs-1.

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Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This study was supported in part by grants to A.S. (Grant-in-Aid for Scientific Research [C] No. 20K06203 by JSPS, and Nos. 20E003, 21E018 and 22K008 by Kurita Water and Environment Foundation). We also thank the Edanz Group (https://jp.edanz.com/ac) for editing the language of a draft of this manuscript.

Funding

This study was supported in part by grants to A.S. (Grant-in-Aid for Scientific Research [C] No. 20K06203 by JSPS, and Nos. 20E003, 21E018 and 22K008 by Kurita Water and Environment Foundation).

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Authors and Affiliations

  1. Department of Natural and Environmental Sciences, School of Science, Academic Assembly, University of Toyama, Toyama, 930-8555, Japan

    Akihiro Sakatoku, Takaya Suzuki, Yuri Tatamiya, Makoto Seki, Daisuke Tanaka & Shogo Nakamura

  2. Graduate School of Bioresources, Mie University, 1577 Kurimamachiya, Tsu, Mie, 514-8507, Japan

    Tadashi Isshiki

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  1. Akihiro Sakatoku
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Contributions

Conceptualization—AS and TI; methodology—AS; formal analysis and investigation—TS, YT and MS; writing of original draft—AS; writing and revision—DT, SN and TI; funding acquisition—AS; supervision—AS and TI.

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Correspondence to Akihiro Sakatoku.

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Sakatoku, A., Suzuki, T., Tatamiya, Y. et al. Development and evaluation of a rapid, specific, and sensitive loop-mediated isothermal amplification assay to detect Tenacibaculum sp. strain pbs-1 associated with black-spot shell disease in Akoya pearl oysters. Arch Microbiol 205, 43 (2023). https://doi.org/10.1007/s00203-022-03384-w

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