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Real-time loop-mediated isothermal amplification assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder

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Abstract

Loop-mediated isothermal amplification (LAMP) assay is a powerful and innovative gene amplification technique that specifically amplifies the target gene under isothermal conditions with a high degree of sensitivity, rapidity and specificity. The major advantage of the LAMP assay is monitoring of amplified products without the requirement of any sophisticated equipment. In the present study a real time LAMP assay was employed for rapid and real time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 2 to 107 spores. DNA was isolated from spiked soil and talcum powder using PBS containing 1% Triton X-100, and heat treatment. Isolated DNA was used as template for LAMP and PCR. LAMP amplification was obtained in 60 min under isothermal condition at 63°C by employing a set of six primers targeting the pag gene of B. anthracis. The detection limit of LAMP assay in soil and talcum powder was found to be as low as 5 spores, compared to 103 spores and 104 spores by PCR in talcum powder and soil, respectively. The findings suggest that LAMP is a more rapid and sensitive assay than PCR for detecting anthrax spores, additionally the methodology to prepare DNA from spiked samples is simple, rapid and cost effective.

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Acknowledgments

We are thankful to Dr. R. Vijayaraghavan, Director, Defence Research and Development Establishment, Ministry of Defense, Government of India, Dr. PVL Rao Division of Virology, Defence Research and Development Establishment, Gwalior for their support and constant inspiration and providing the necessary facilities for this study. We are also thankful to Professor R. M. Agarwal, Jiwaji University, Gwalior for his valuable support to carry out this work.

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Authors and Affiliations

  1. Division of High Containment Facility, Defence Research & Development Establishment (DRDE), Jhansi Road, Gwalior, 474002, Madhya Pradesh, India

    Neha Jain, S. Merwyn, G. P. Rai & G. S. Agarwal

  2. Division of Virology, Defence Research & Development Establishment, Gwalior, 474002, India

    Jyoti S. Kumar & M. M. Parida

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  1. Neha Jain
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  2. Jyoti S. Kumar
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  3. M. M. Parida
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  4. S. Merwyn
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  5. G. P. Rai
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  6. G. S. Agarwal
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Correspondence to G. S. Agarwal.

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Jain, N., Kumar, J.S., Parida, M.M. et al. Real-time loop-mediated isothermal amplification assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder. World J Microbiol Biotechnol 27, 1407–1413 (2011). https://doi.org/10.1007/s11274-010-0592-3

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  • DOI: https://doi.org/10.1007/s11274-010-0592-3

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