Real-time PCR system targeting a chromosomal marker specific for Bacillus anthracis

Effects of <i>Bacillus anthracis</i> hydrophobicity and induction of host cell death on sample collection from environmental surfaces

“…Reaction assays were run on an ABI 7900 HT sequence detection system (Applied Biosystems) using the following conditions: 94 C for 10 m and 40 cycles of 94 C for 15 s and 60 C for 1 min. For B. anthracis, the chromosomal marker gene locus BA_5345 was used to detect pathogen DNA by TaqMan assay: Primer 1: dhp61_183-113F 5 cgt aag gac aat aaa agc cgt tgt 3 , Primer 2: dhp61_183_208R 5 cga tac aga cat tta ttg gga act aca c 3 , and Probe: dhp61_183-143T 6FAM-tgc aat cga tga gct aat gaa caa tga ccc t-MGB (Antwerpen et al, 2008). B. anthracis DNA standards (10-fold dilutions of DNA from 1 ng to 10 fg) were prepared and analyzed by qPCR in parallel as described above.…”

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

“…These assays target the markers BA5345, 21,65 PL3, 47 and BA5357, 46 respectively. Three of these assays are based on hydrolysis probe ("TaqMan assay"); the fourth uses SYBR Green chemistry.…”

“…Six published PCR-assays targeting different B. anthracis chromosomal markers were evaluated in vitro. The most specific methods according to in silico analysis 21,46,47 were compared with the assays recommended by the WHO 40,44 and a single assay targeting BA813. 35 Ninety blinded DNA samples were exchanged between partners and an IAC was distributed.…”

“…[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.…”

Injectional Anthrax in Heroin Users, Europe, 2000–2012

“…Molecular phylogenetic methods were used to determine the genetic relatedness of these strains with Ba4599 (8,9). Genotyping results using canonical single nucleotide polymorphisms (SNPs) (Figure) (2,4) placed all of these strains along branch A.Br.008 within the Trans-Eurasian group of B. anthracis (10).…”