Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates

Abstract

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five polymorphisms differed by the presence of two to six copies of the 12-bp tandem repeat 5-CAATATCAACAA-3. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations.

Rapid genotypic detection ofBacillus anthracisand theBacillus cereusgroup by multiplex real-time PCR melting curve analysis

“…anthracis is a highly fatal infectious agent in animals and humans and therefore its early and unambiguous diagnostic detection is essential for successful treatment and disease prevention. There have been many efforts to utilize rapid DNA-based detection methods, such as PCR [22][23][24][25][26][27][28][29][30][31][32][33], to replace time-consuming biochemical or culture-based diagnostic tests [34]. PCR-based methods can readily differentiate vaccine or fully virulent B. anthracis plasmid genotypes [22][23][24]26,35].…”

“…PCR methods developed for detection of the B. anthracis chromosome have suffered from lack of assay specificity; Ba813 [25,26,51], vrrA gene [27][28][29], gyrase B gene (gyrB) [30], SG-850 [31], the b subunit of RNA Table 1 Bacterial strains used in this study and their DNA-based assay response (manuscript in preparation)…”

Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysis

“…The probes that experimentally gave the highest and most specific signals in preliminary tests with purified DNA were used for this study. Bacillus species probes were designed containing two to five copies of the variable number tandem repeat (VNTR) regions of B. anthracis (Jackson et al 1997) in an attempt to differentiate strains of B. anthracis according to their VNTR profile. Probes complementary to the ITS regions of 16S-23S ribosomal DNA of B. anthracis and a homologous region in B. anthracis, B. cereus and B. thuringiensis were also designed.…”

The native virulence plasmid combination affects the segregational stability of a theta-replicating shuttle vector in Bacillus anthracis var. New Hampshire

“…The remaining pAEX-5E DNA was purified using QIAquick Gel Extraction Kit (Qiagen Ltd, Crawley, UK) and labelled using the ECL random prime labelling kit (Amersham Life Science, Little Chalfont, UK) according to the manufacturer's instructions. Total chromosomal and plasmid DNA was extracted from derivatives of B. anthracis using the method of Jackson et al (1997). DNA (approximately 1 mg) was digested overnight at 37°C with 70 units of enzyme in the appropriate buffer, according to the manufacturer's instructions (NBL Gene Sciences Ltd, Cramlington, UK).…”