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

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.