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

Abstract

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.

FIG. 1

Alignment of the nucleotide sequences from 10 representative Bacillus strains using Clustal W (32). The strains are the following: Bc 27877, Bacillus cereus 27877; Bc 23261, Bacillus cereus 23261; Bt 35646, Bacillus thuringiensis 35646; Ba Vollum, Bacillus anthracis Vollum; Ba A74, Bacillus anthracis A74; Ba Sterne, Bacillus anthracis Sterne; Ba813_11, Bacillus sp. strain Ba813_11; Ba813_12, Bacillus sp. strain Ba813_12; Bt B8, Bacillus thuringiensis BtB8; Bs 6051, Bacillus subtilis 6051. The locations of primers and probes are shown (F, fluorescein; Cy5, cyanine 5; P, phosphate group); the presence of an asterisk denotes a mismatch, a dash indicates identity with the consensus sequence, and nucleotide letters indicate positions showing polymorphism.

FIG. 2

Results of the FRET-PCR assay using genomic DNA. (A) Fluorescence ratio (F2/F1) is plotted against the number of PCR cycles. The samples are the following: 1, □, negative control (no DNA); 2, ▵, Bacillus anthracis A74; 3, ○, Bacillus anthracis Sterne; 4, , Bacillus sp. strain Ba813_11; 5, +, Bacillus anthracis Vollum; 6, ●, Bacillus cereus 27877; 7, ◊, Bacillus cereus 23261; 8, −, Bacillus sp. strain Ba813_12; 9, ▴, Bacillus thuringiensis BtB8. (B) Gel electrophoresis of the PCR products. Lanes, M: 100 bp DNA ladder; 1, negative control (no DNA); samples 2 to 9 are the same as in panel A.

FIG. 3

Results of the FRET-PCR assay using crude vegetative cell lysates. The fluorescence ratio (F2/F1) is plotted against the number of PCR cycles. The sample are the following: ▴, negative control (no DNA); ■, Bacillus anthracis AC3; ▵, Bacillus anthracis 7702; ×, Bacillus anthracis ΔUM-2311; , Bacillus anthracis A74; ●, Bacillus anthracis 0074; +, Bacillus anthracis Texas 0077; −, Bacillus anthracis ΔANR-1099; ○, Bacillus anthracis 7700; ◊, Bacillus anthracis A58; □, Bacillus cereus 14579; ♦, Bacillus thuringiensis 10792.