Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/ microliters and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between C(T) value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.