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.Bacillus anthracis is a causal agent of anthrax, a serious and often fatal infection of livestock and humans. It is considered one of the most effective biological weapons of mass destruction because of its highly pathogenic nature and spore-forming capability and has attracted attention due to its potential use as a biological warfare agent (2). This bacterium can infect humans by cutaneous, gastrointestinal, or respiratory routes. The standard laboratory method of identification takes advantage of the lytic nature of the B. anthracis-specific gamma bacteriophage (9). Anthrax bacilli are often distinguished on the basis of time-consuming morphological or phenotypic characteristics, such as gram-positive staining, spore-forming capability, nonhemolytic reaction on sheep blood agar, sensitivity to penicillin, nonmotile nature, and inability to ferment salicin (11). B. anthracis is distinguished from the other members of the closely related Bacillus cereus group of bacteria by the presence of the toxin-encoding pXO1 (19, 24) and capsule-encoding pXO2 plasmids (14,23,34). Both plasmids are needed for virulence; thus, the absence of either plasmid results in attenuation.B. anthracis, Bacillus thuringiensis, B. cereus, and Bacillus mycoides, are members of the B. cereus group of bacilli. These closely related bacteria are pathogens of mammals (B. anthracis and B. cereus) and insects (B. thuringiensis). The B. cereus group is one of the most taxonomically ambiguous group of bacilli (27). In fact, DNA-DNA hybridization (30) and pulsedfield gel electrophoresis (15) have shown great homology among B. anthracis, B. thuringiensis, and B. cereus. A recent multilocus enzyme electrophoresis study has concluded that the members of this group belong to one species (16)....

Selection and Characterization of Single Domain Antibodies Specific for Bacillus anthracis Spore Proteins

“…Though discrimination of the closely related bacteria of the B. cereus group is possible with assays that utilize PCR [4][5][6], these techniques frequently require laboratory based equipment and skilled technicians. Immunoassays, such as lateral flow assays, offer advantages over PCR-based techniques in their simplicity, portability, and low cost allowing them to readily be deployed to the field.…”

Genetic distribution of 295 Bacillus cereus group members based on adk-screening in combination with MLST (Multilocus Sequence Typing) used for validating a primer targeting a chromosomal locus in B. anthracis

“…Several chromosomally derived PCRprimers for identifying B. anthracis have been published, targeted against BA813 (Ramisse et al, 1996), saspB (Hoffmaster et al, 2002), rpoB (Qi et al, 2001), gyrA (Hurtle et al, 2004), a fragment crossing a hypothetical protein and a alpha/betahydrolase encoding genes (Bode et al, 2004) and plcR (Easterday et al, 2005).…”

In silico and in vitro evaluation of PCR-based assays for the detection ofBacillus anthracischromosomal signature sequences

“…[13][14][15] Finally, a few single nucleotide polymorphisms (SNP) have also been considered for PCR markers. Target genes include rpoB, 24,[48][49][50][51] gyrA, 25,52,53 gyrB, 54,55 plc, 20,23,53,56 purA, 57 and the 16S-23S rDNA internal spacer sequences. [58][59][60] But, so far, only the nonsense mutation in the global regulator PlcR, which controls the transcription of secreted virulence factors in B. cereus and B. thuringiensis, have proved to be truly unique to B. anthracis strains.…”

“…16,20,59 False-positive signals have sometimes been recorded with closely related strains of the B. cereus group using the other published SNPs. 24,49,52,59,[61][62][63] In silico analysis About a hundred sequences corresponding to all primers and probes currently published were compiled and compared using the primer alignment function of the Gegenees software (www. gegenees.org).…”

“…Small alterations in these conditions can result in the loss of specificity, especially with hydrolysis probes, i.e., TaqMan chemistry. 18,[23][24][25] To evaluate the wide range of PCR methods used in laboratories for B. anthracis identification, a computer-based comparative analysis of more than 300 PCR-target sequences reported in the literature was conducted. All sequences were compared against all publicly available Bacillus genomes and sorted for specificity.…”