A simple and sensitive method for detection of Bacillus anthracis by loop-mediated isothermal amplification

Authors

  • 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
    3.  Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
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  • 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
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  • A. Fukuma,

    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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  • K. Kawamoto,

    1.  Research Center for Animal Hygiene and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan
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  • N. Kamo,

    1.  Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
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  • S.-I. Makino,

    1.  Research Center for Animal Hygiene and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, 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, Fifth Biology Section for Microbiology, 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 rapid and simple system for detection of Bacillus anthracis using a loop-mediated isothermal amplification (LAMP) method and determine the suitability of LAMP for rapid identification of B. anthracis infection.

Methods and Results:  A specific LAMP assay targeting unique gene sequences in the bacterial chromosome and two virulence plasmids, pXO1 and pXO2, was designed. With this assay, it was possible to detect more than 10 fg of bacterial DNA per reaction and obtain results within 30–40 min under isothermal conditions at 63°C. No cross-reactivity was observed among Bacillus cereus group and other Bacillus species. Furthermore, in tests using blood specimens from mice inoculated intranasally with B. anthracis spores, the sensitivity of the LAMP assay following DNA extraction methods using a Qiagen DNeasy kit or boiling protocol was examined. Samples prepared by both methods showed almost equivalent sensitivities in LAMP assay. The detection limit was 3·6 CFU per test.

Conclusions:  The LAMP assay is a simple, rapid and sensitive method for detecting B. anthracis.

Significance and Impact of the Study:  The LAMP assay combined with boiling extraction could be used as a simple diagnostic method for identification of B. anthracis infection.

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