Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification

Authors

  • T. Sakuma,

    1.  First Department of Forensic Science, National Research Institute of Police Science, Kashiwa, Japan
    2.  CREST, Japan Science and Technology Agency, Saitama, Japan
    3.  Graduate School of Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan
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  • Y. Kurosaki,

    1.  First Department of Forensic Science, National Research Institute of Police Science, Kashiwa, Japan
    2.  CREST, Japan Science and Technology Agency, Saitama, Japan
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  • Y. Fujinami,

    1.  First Department of Forensic Science, National Research Institute of Police Science, Kashiwa, Japan
    2.  CREST, Japan Science and Technology Agency, Saitama, Japan
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  • T. Takizawa,

    1.  Graduate School of Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan
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  • J. Yasuda

    1.  First Department of Forensic Science, National Research Institute of Police Science, Kashiwa, Japan
    2.  CREST, Japan Science and Technology Agency, Saitama, Japan
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Jiro Yasuda, First Department of Forensic Science, National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa 277-0882, Japan. E-mail: yasuda@nrips.go.jp

Abstract

Aims:  To develop a convenient and rapid detection method for toxigenic Clostridium botulinum types A and B using a loop-mediated isothermal amplification (LAMP) method.

Methods and results:  The LAMP primer sets for the type A or B botulinum neurotoxin gene, BoNT/A or BoNT/B, were designed. To determine the specificity of the LAMP assay, a total of 14 C. botulinum strains and 17 other Clostridium strains were tested. The assays for the BoNT/A or BoNT/B gene detected only type A or B C. botulinum strains, respectively, but not other types of C. botulinum or strains of other Clostridium species. Using purified chromosomal DNA, the sensitivity of LAMP for the BoNT/A or BoNT/B gene was 1 pg or 10 pg of DNA per assay, respectively. The assay times needed to detect 1 ng of DNA were only 23 and 22 min for types A and B, respectively. In food samples, the detection limit per reaction was one cell for type A and 10 cells for type B.

Conclusions:  The LAMP is a sensitive, specific and rapid detection method for C. botulinum types A and B.

Significance and Impact of the Study:  The LAMP assay would be useful for detection of C. botulinum in environmental samples.

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