Multiple-Locus Variable-Number Tandem Repeat Analysis Reveals Genetic Relationships within Bacillus anthracis

Abstract

Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC 1 , vrrC 2 , vrrB 1 , vrrB 2 , and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (vrrA). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.

Microevolution and history of the plague bacillus, Yersinia pestis

“…Such inaccurate branch orders are method-specific and can be recognized by comparing the results from independent methods with different clock rates. We therefore investigated the evolutionary history of Y. pestis by three independent high-resolution methods that have been applied to monomorphic species: synonymous SNPs (sSNPs) defined by genome scanning (22,23), MLVA (24,25), and screening for the presence of IS100 at defined locations (4).…”

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

“…[13][14][15] The first DNA signatures that were developed for anthrax PCR detection methods independently of plasmids occurrence were DNA fragments used to genotype B. anthracis. They include the vrrA marker, [26][27][28] the AC-390 gene, 29 and the SG-850/749 fragment. 30 These genetic markers provide limited specificity and require additional timeconsuming and labor-intensive post-PCR analysis steps.…”

Microbial Forensics: DNA Fingerprinting of Bacillus anthracis (Anthrax)

“…Although the numbers of such sites in bacterial genomes vary considerably, VNTR markers have been used in many systems to provide the highest level of resolution for strain identification for several bacteria, including B. anthracis, Y. pestis, Brucella sp., and E. coli O157 (10,(29)(30)(31). And as in the FBI CODIS STR marker system for humans, when the highest degree of resolution was required, a multiple-locus VNTR analysis (MLVA) system was adapted for B. anthracis (30).…”