Real-time polymerase chain reaction assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder
2012
Jain, Neha | Merwyn, S. | Rai, G. P. | Agarwal, G. S.
Real-time polymerase chain reaction (real-time PCR) is a laboratory technique based on PCR. This technique is able to detect sequence-specific PCR products as they accumulate in “real time” during the PCR amplification, and also to quantify the number of substrates present in the initial PCR mixture before amplification begins. In the present study, real-time PCR assay was employed for rapid and real-time detection of Bacillus anthracis spores spiked in 0.1Â g of soil and talcum powder ranging from 5 to 107 spores. DNA was isolated from spiked soil and talcum powder, using PBS containing 1Â % Triton-X-100, followed by heat treatment. The isolated DNA was used as template for real-time PCR and PCR. Real-time PCR amplification was obtained in 60Â min under the annealing condition at 60°C by employing primers targeting the pag gene of B. anthracis. In the present study, the detection limit of real-time PCR assay in soil was 103 spores and102 spores in talcum powder, respectively, whereas PCR could detect 104 spores in soil and 103 spores in talcum powder, respectively.
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