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Applied and Environmental Microbiology, August 2001, p. 3720-3727, Vol. 67, No. 8
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.8.3720-3727.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Utilization of the rpoB Gene as a Specific Chromosomal Marker for Real-Time PCR Detection of Bacillus anthracis

Yuan Qi,dagger Guy Patra, Xudong Liang, Leanne E. Williams, Sharon Rose, Rajendra J. Redkar, and Vito G. DelVecchio*

Institute of Molecular Biology and Medicine, University of Scranton, Scranton, Pennsylvania 18510

Received 20 February 2001/Accepted 5 June 2001

The potential use of Bacillus anthracis as a weapon of mass destruction poses 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 1 of the rpoB gene was sequenced from 36 Bacillus strains, including 16 B. anthracis strains and 20 other related bacilli, and four nucleotides specific for B. anthracis were identified. PCR primers were selected so that two B. anthracis-specific nucleotides were at their 3' ends, whereas the remaining bases were specific to the probe region. This format permitted the PCR reactions to be performed on a LightCycler via fluorescence resonance energy transfer (FRET). The assay was found to be specific for 144 B. anthracis strains from different geographical locations and did not cross-react with other related bacilli (175 strains), with the exception 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.

* Corresponding author. Mailing address: University of Scranton, Institute of Molecular Biology and Medicine, Scranton, PA 18510-4625. Phone: (570) 941-4817. Fax: (570) 941-6229. E-mail: vimbm{at}aol.com.

dagger Present address: Biomedical Engineering Graduate Program, University of Texas, Southwestern Medical Center at Dallas, Dallas, TX 75390-9178.

Applied and Environmental Microbiology, August 2001, p. 3720-3727, Vol. 67, No. 8
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.8.3720-3727.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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