Real-Time PCR Assay for a Unique Chromosomal Sequence of Bacillus anthracis

Abstract

Real-time PCR has become an important method for the rapid identification of Bacillus anthracis since the 2001 anthrax mailings. Most real-time PCR assays for B. anthracis have been developed to detect virulence genes located on the pXO1 and pXO2 plasmids. In contrast, only two published chromosomal targets exist, the rpoB gene and the gyrA gene. In the present study, subtraction-hybridization with a plasmid-cured B. anthracis tester strain and a Bacillus cereus driver was used to find a unique chromosomal sequence. By targeting this region, a real-time assay was developed with the Ruggedized Advanced Pathogen Identification Device. Further testing has revealed that the assay has 100% sensitivity and 100% specificity, with a limit of detection of 50 fg of DNA. The results of a search for sequences with homology with the BLAST program demonstrated significant alignment to the recently published B. anthracis Ames strain, while an inquiry for protein sequence similarities indicated homology with an abhydrolase from B. anthracis strain A2012. The importance of this chromosomal assay will be to verify the presence of B. anthracis independently of plasmid occurrence.Bacillus anthracis is a spore-forming gram-positive bacterium well known for its recent use as a bioterrorist agent. Identification of B. anthracis can be done clinically by Gram stain, colony morphology, and various biochemical tests (19). However, these methods are time-consuming, and more rapid tests, such as PCR, have been used to detect B. anthracis in clinical samples (20). Real-time PCR is preferred over conventional PCR methods for the identification of organisms because it is fast, is less labor-intensive, and adds the specificity of a probe. While real-time PCR assays have been used to identify B. anthracis on the basis of the virulence genes associated with the toxin-encoding plasmid (pXO1) and the capsule-encoding plasmid (pXO2) (11,20,22), a reliable chromosomal assay has not been developed. Chromosomal assays can be valuable tools when they are used in conjunction with virulence gene assays because they provide information on the genetic contexts of the pXO1 and pXO2 plasmids. While the chromosomal assays may not prove useful as initial screening assays, they can certainly have a significant role in confirmatory testing as part of an integrated diagnostic approach.Past attempts to develop a chromosomal real-time PCR assay have failed due to the close genetic relationship of Bacillus species. B. anthracis, Bacillus cereus, and Bacillus thuringiensis have very little variability and are genetically indistinguishable by multilocus enzyme electrophoresis (10). Recent work by repetitive PCR has shown that the previously listed species of Bacillus, as well as Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis, do have some genetic differences (3). Real-time PCR assays based on the chromosomal rpoB and gyrA genes of B. anthracis have been developed (5, 13, 25). However, these assays are based on single-nucleotide differences...

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).…”

Development of Three Real-Time PCR assays to Detect Bacillus anthracis and Assessment of Diagnostic Utility

“…Although PCR has a theoretical limit of detection of 1 target per assay, in reality the stochastic distribution of DNA at low concentrations [15] makes it impossible to define a reproducible Limit of Detection below 3 GEs [16]. Our assays would appear to have reproducible limits of detection somewhere in the range of 100 fg-17 fg (17.5 GEs to 3GEs) per PCR, consistent with other B. anthracis PCRs [17].…”

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

“…30 These genetic markers provide limited specificity and require additional timeconsuming and labor-intensive post-PCR analysis steps. Other areas of the chromosome have also been investigated as potential DNA-targets for identification purposes, including the so-called BA813 [31][32][33][34][35][36][37][38] and BA5510 sequences, 19 genes bclB, 39 sap, 40,41 saspB, 5,42 and sspE, 22,43 the B-type small acid-soluble spore protein gene (SASP), 44 a glycosyltransferase group 1 family protein, 45 a protein showing similarities with an abhydrolase, 18 and several DNA loci located on prophage regions, 17 i.e., BA5345, 21 BA5357, 46 and PL3. 47 Although most of these regions have been claimed to be anthrax-specific, B. cereus strains sometimes yield false positive results.…”

“…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.…”

“…[13][14][15] Few chromosomal sequences that provide sufficient polymorphism to unambiguously distinguish B. anthracis from its near neighbors have been identified. 14,[16][17][18][19][20][21][22] Some of these assays rely upon single-nucleotide differences for discrimination and are therefore sensitive to assay conditions and PCR cycling parameters. Small alterations in these conditions can result in the loss of specificity, especially with hydrolysis probes, i.e., TaqMan chemistry.…”