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Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue

    Motoki Goto

    Section of Animal Research, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

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    ,
    Eiichi Honda

    Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan

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    ,
    Atsuo Ogura

    Section of Animal Research, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

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    ,
    Akio Nomoto

    Department of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

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    &
    Ken-Ichi Hanaki

    *Address correspondence to Ken-Ichi Hanaki, D.V.M., Ph.D., Section of Animal Research, CDBIM, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan. e-mail:

    E-mail Address: khanaki@m.utokyo.ac.jp

    Section of Animal Research, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

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    Published Online:25 Apr 2018https://doi.org/10.2144/000113072

    Abstract

    Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, enables the synthesis of larger amounts of both DNA and a visible byproduct—namely, magnesium pyrophosphate—without thermal cycling. A positive reaction is indicated by the turbidity of the reaction solution or the color change after adding an intercalating dye to the reaction solution, but the use of such dyes has certain limitations. Hydroxy naphthol blue (HNB), a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions, was used for a new colorimetric assay of the LAMP reaction. Preaddition of 120 µM HNB to the LAMP reaction solution did not inhibit amplification efficiency. A positive reaction is indicated by a color change from violet to sky blue. The LAMP reaction with HNB could also be carried out in a 96-well microplate, and the reaction could be measured at 650 nm with a microplate reader. The colorimetric LAMP method using HNB would be helpful for high-throughput DNA and RNA detection.

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