Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis

The potential use of Bacillus anthracis as a weapon of mass destruction pos es a threat to humans, domesticated animals, and wildlife and necessitates the need for a rapid and highly specific detection assay. We have developed a real-time PCR-based assay for the specific detection of B. anthracis by taking advantage of the unique nucleotide sequence of the B. anthracis rpoB gene. Variable region I of the rpoB gene was sequenced from 36 Bacillus st rains, including 16 B. anthracis strains and 20 other related bacilli, and four nucleotides specific for B. anthracis were identified. PCR primers wer e selected so that two B. anthracis-specific nucleotides were at their 3 ' ends, whereas the remaining bases were specific to the probe region. This f ormat permitted the PCR reactions to be performed on a LightCycler via fluo rescence resonance energy transfer (FRET). The assay was found to be specif ic for 144 B. anthracis strains from different geographical locations and d id not cross-react with other related bacilli (175 strains), with the excep tion of one strain. The PCR assay can be performed on isolated DNA as well as crude vegetative cell lysates in less than 1 h. Therefore, the rpoB-FRET assay could be used as a new chromosomal marker for rapid detection of B. anthracis.