Strain-Specific Single-Nucleotide Polymorphism Assays for the Bacillus anthracis Ames Strain

Abstract

Highly precise diagnostics and forensic assays can be developed through a combination of evolutionary analysis and the exhaustive examination of genomic sequences. In Bacillus anthracis, whole-genome sequencing efforts revealed ca. 3,500 single-nucleotide polymorphisms (SNPs) among eight different strains and evolutionary analysis provides the identification of canonical SNPs. We have previously shown that SNPs are highly evolutionarily stable, and the clonal nature of B. anthracis makes them ideal signatures for subtyping this pathogen. Here we identified SNPs that define the lineage of B. anthracis that contains the Ames strain, the strain used in the 2001 bioterrorist attacks in the United States. Sequencing and real-time PCR were used to validate these SNPs across B. anthracis strains, including (i) 88 globally and genetically diverse isolates; (ii) isolates that were shown to be genetic relatives of the Ames strain by multiple-locus variable number tandem repeat analysis (MLVA); and (iii) several different lab stocks of the Ames strain, including a clinical isolate from the 2001 letter attack. Six SNPs were found to be highly specific for the Ames strain; four on the chromosome, one on the pX01 plasmid, and one on the pX02 plasmid. All six SNPs differentiated the B. anthracis Ames strain from the 88 unique B. anthracis strains, while five of the six separated Ames from its close genetic relatives. The use of these SNPs coupled with real-time PCR allows specific and sensitive (<100 fg of template DNA) identification of the Ames strain. This evolutionary and genomics-based approach provides an effective means for the discovery of strain-specific SNPs in B. anthracis.The 2001 anthrax letter attack illustrated the "real-world" efficacy of Bacillus anthracis as a bioterror agent. Forensic and epidemiological analysis of clinical samples and weaponized spores from the letter attack included the identification of the B. anthracis strain as Ames (3,8,17). This was initially accomplished using multiple-locus variable number tandem repeat analysis (MLVA) (3,6,8), which was one of the few technical approaches possible for this highly monomorphic pathogen (5). Identifying the strain and establishing its identity in attack locations were important in linking the dispersed anthrax cases and suggesting a possible source for the weaponized material. During the anthrax attack crisis, diagnostic speed, specificity, and sensitivity were often limiting factors, necessitating extraordinary efforts by public health officials and forensic labs (3). Further advancement of molecular diagnostics allows for more-efficient responses in disease outbreaks, whether natural or bioterrorist mediated.Single-nucleotide polymorphisms (SNPs) are increasingly recognized as important markers for detecting and subtyping bacterial pathogens, including B. anthracis (1,2,4,8,16,18,19). Recent comparative full-genome sequencing allowed the discovery of about 3,500 SNPs among eight strains of B. anthracis (15, 17; J. Ravel, unpublished data) and repre...

Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

“…A nested hierarchical approach involving SNP analysis to define the major branches, followed by analysis using more rapidly evolving VNTR markers, will generate unambiguous differentiation of isolates into major clades, with maximum resolution at the individual isolate level (19). SNP-based differentiation of clades can then be incorporated into real-time PCR assays, providing a quick method for determining specific groupings (23,32,34).…”

Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation

“…For example, although current genotyping systems are able to differentiate strains of B. anthracis, they are unable to distinguish rare subpopulations within individual cultures. B. anthracis is highly monomorphic, and accurate genotyping relies on a combination of canonical SNPs and multiple-locus variable-number tandem repeat analyses (1,4,(24)(25)(26). We started with the naïve approach that a single reference genome and sample would be sufficient for comparison.…”

Single Nucleotide Polymorphism Typing ofBacillus anthracisfrom Sverdlovsk Tissue

“…This SNP was not found in any of the original 27 diverse B. anthracis isolates (5) or in a recent pagA gene sequencing analysis of 124 archival B. anthracis samples from the Centers for Disease Control and Prevention (13). The status of the new pagA 981 site was also analyzed in a panel of isolates containing 89 diverse multiple-locus, variable-number, tandem-repeat analysis genotypes and an additional 154 A.Br.008/009 subgroup (Eurasian) isolates by using a custom made real-time PCR assay: TaqMan Minor Groove Binding probes and primers for SSNPs (Applied Biosystems) (14). These analyses indicate that the pagA 981 A-T transversion represents a rare SNP allele found in only 3 Eurasian isolates and the Sverdlovsk 7.RA93.15.15 sample.…”