Use of single nucleotide polymorphisms in the plcR gene for specific identification of Bacillus anthracis

Abstract

A TaqMan-minor groove binding assay designed around a nonsense mutation in the plcR gene was used to genotype Bacillus anthracis, B. cereus, and B. thuringiensis isolates. The assay differentiated B. anthracis from these genetic near-neighbors and determined that the nonsense mutation is ubiquitous across 89 globally and genetically diverse B. anthracis strains.

FIG. 1.

Sequence alignment of plcR gene fragments from B. anthracis and genetic near-neighbors. The numbered lines indicate the sequences of (line 1) the primers and probes used in the assay; (line 2) B. anthracis strains Ames, Vollum, A2012, A1055, AUS94, CNEVA9066, Kruger, Sterne, and WNA6153; (line 3) B. cereus strains 3A (GenBank accession no. AY785766) and S2-8 and B. thuringensis strain HD1011; (line 4) B. thuringensis strain 97-27 (AY785771); (line 5) near-neighbor strain TET 2b-3; (line 6) B. cereus strain AH-527 (AY785767); (line 7) B. cereus strain D17 (AY785768) and B. thuringensis strains HD682, HD571, and HD44; (line 8) B. cereus strains F3502/72 (AY785769) and R6; (line 9) B. cereus strain F2-1 (AY785770); and (line 10) B. cereus strains R4 and ATCC 33018 and B. thuringensis strain HD1012 (AY785772). Light shading indicates areas of polymorphism that are detected in the assay. Darker shading indicates nucleotide differences between near-neighbors and B. anthracis. * indicates the nonsense mutation.

FIG. 2.

Results of triplicate analysis of 10-fold serial dilutions of DNA from B. anthracis strain A0442. The average cycle threshold values across the three replicates were as follows: 100 pg, 21.4; 10 pg, 24.7; 1 pg, 28.2; 100 fg, 31.2; 10 fg, 34.3. The average cycle threshold values for strains A0488 and A0462 were similar (data not shown), although amplification at the 10-fg level was not consistent.