Rapid identification of Bacillus anthracis by real-time PCR with dual hybridization probes in environmental swabs

In the present study, we report the development of a real-time PCR assay for the identification of Bacillus anthracis, based on the amplification of a unique chromosomal marker, the E4 sequence, with dual hybridization probes. The assay was evaluated using a panel of ten B. anthracis strains, two B. anthracis isolates from human clinical samples, 12 B. anthracis environmental swabs and 40 non-B. anthracis strains. All 12 B. anthracis strains and clinical isolates were correctly detected, and the method did not show cross-reactions with other microorganisms. Likewise, the E4 sequence was not found in those strains of B. thuringiensis and B. cereus closely related (homology > 90%) to B. anthracis by computer analysis. On the other hand, this molecular assay showed a high analytical sensitivity, 3.5 genome equivalents per reaction at 95% probability. Furthermore, the real-time PCR assay allowed sequence-specific detection of the amplicon (melting peak with a Tm of 63.5 degrees C +/- 0.5 degrees C) without post-amplification procedures, which offers an additional advantage over other qPCR assays for B. anthracis detection. Finally, the performance of the method was successfully evaluated in 12 environmental samples. In summary, we have developed a rapid and specific method for the molecular identification of Bacillus anthracis in environmental samples.