A multiplex real-time PCR for identifying and differentiating B. anthracis virulent types

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

“…Other areas of the chromosome have also been investigated as potential DNA-targets for identification purposes, including the so-called BA813 [31][32][33][34][35][36][37][38] and BA5510 sequences, 19 genes bclB, 39 sap, 40,41 saspB, 5,42 and sspE, 22,43 the B-type small acid-soluble spore protein gene (SASP), 44 a glycosyltransferase group 1 family protein, 45 a protein showing similarities with an abhydrolase, 18 and several DNA loci located on prophage regions, 17 i.e., BA5345, 21 BA5357, 46 and PL3. 47 Although most of these regions have been claimed to be anthrax-specific, B. cereus strains sometimes yield false positive results. [13][14][15] Finally, a few single nucleotide polymorphisms (SNP) have also been considered for PCR markers.…”

“…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.…”

“…44 Results of the ring trial confirmed the results obtained in the in silico analysis ( Table 4). The three assays with highest in silico specificity (BA5345, 21 PL3, 47 and BA5357 46 ) all performed well in the ring trial, with diagnostic sensitivity and specificity values close to 1 ( Table 5). Furthermore, these assays were found to be robust and provided consistent results between laboratories (kappa values of 0.9-1.0).…”

“…47 Serial dilutions of genomic DNA from B. anthracis strain 17JB were tested to determine the lowest concentration of DNA that could be detected at 95% probability. The detection limit (LOD PCR at 95% confidence interval) was found to be 2 genome equivalents.…”

“…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.…”