Specific detection of bacillus anthracis using a TaqMan mismatch amplification mutation assay

Single nucleotide polymorphisms (SNPs) are increasingly recognized as important diagnostic markers for the detection and differentiation of Bacillus anthracis. The use of SNP markers for identifying B. anthracis DNA in environmental samples containing genetically similar bacteria requires the ability to amplify and detect DNA with single nucleotide specificity. We designed a TaqMan mismatch amplification mutation assay (TaqMAMA) around a SNP in the plcR gene of B. anthracis. The assay permits specific, low-level detection (25 fg DNA) of this B. anthracis-specific SNP, even in the presence of environmental DNA extracts containing a 20,000-fold excess of the alternate allele. We anticipate that the ability to selectively amplify and detect low copy number DNAs with single nucleotide specificity will represent a valuable tool in the arena of biodefense and microbial forensics.     

High resolution genotyping of Bacillus anthracis outbreak strains using four highly mutable single nucleotide repeat markers

Aims: Bacillus anthracis is a genetically monomorphic bacterium with little diversity to be expected during an outbreak. This study used more rapidly evolving genetic markers on outbreak samples to ascertain genetic diversity. Methods and Results: Forty‐seven isolates from a B. anthracis outbreak during the summer of 2005 in South Dakota were analysed using single nucleotide polymorphisms (SNP) and multi‐locus VNTR analysis (MLVA). Results indicated that all of the outbreak strains belonged to a single clonal lineage. However, analysis of four single nucleotide repeat (SNR) markers resolved these isolates into six distinct genotypes providing insights into disease transmission. Conclusions: Strain determination of unknown B. anthracis samples can be ascertained by SNP and MLVA markers. However, comparison of many samples obtained during an outbreak will require markers with higher rates of mutation to ascertain genetic diversity. Significance and Impact of the Study: SNR4 analysis allowed discrimination of closely related B. anthracis isolates and epidemiological tracking of the outbreak. When used in conjunction with other genotyping schemes that allow broad genetic relationships to be determined, SNR markers are powerful tools for detailed tracking of natural B. anthracis outbreaks and could also prove useful in forensic investigations.     

A high resolution four-locus multiplex single nucleotide repeat (SNR) genotyping system in Bacillus anthracis

The allelic identities of Single Nucleotide Repeat (SNR) markers in Bacillus anthracis are typically ascertained by DNA sequencing through the direct repeat. Here we describe a reproducible method for genotyping closely related isolates by using four SNR loci in a multiplex-PCR capillary electrophoresis system amenable to high-throughput analysis.     

Global genetic population structure of Bacillus anthracis

Anthrax, caused by the bacterium Bacillus anthracis, is a disease of historical and current importance that is found throughout the world. The basis of its historical transmission is anecdotal and its true global population structure has remained largely cryptic. Seven diverse B. anthracis strains were whole-genome sequenced to identify rare single nucleotide polymorphisms (SNPs), followed by phylogenetic reconstruction of these characters onto an evolutionary model. This analysis identified SNPs that define the major clonal lineages within the species. These SNPs, in concert with 15 variable number tandem repeat (VNTR) markers, were used to subtype a collection of 1,033 B. anthracis isolates from 42 countries to create an extensive genotype data set. These analyses subdivided the isolates into three previously recognized major lineages (A, B, and C), with further subdivision into 12 clonal sub-lineages or sub-groups and, finally, 221 unique MLVA15 genotypes. This rare genomic variation was used to document the evolutionary progression of B. anthracis and to establish global patterns of diversity. Isolates in the A lineage are widely dispersed globally, whereas the B and C lineages occur on more restricted spatial scales. Molecular clock models based upon genome-wide synonymous substitutions indicate there was a massive radiation of the A lineage that occurred in the mid-Holocene (3,064-6,127 ybp). On more recent temporal scales, the global population structure of B. anthracis reflects colonial-era importation of specific genotypes from the Old World into the New World, as well as the repeated industrial importation of diverse genotypes into developed countries via spore-contaminated animal products. These findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease. Finally, the value of global genotypic analysis for investigating bioterrorist-mediated outbreaks of anthrax is demonstrated.