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. 2004 Dec;42(12):5825-31.
doi: 10.1128/JCM.42.12.5825-5831.2004.

Real-time PCR assay for a unique chromosomal sequence of Bacillus anthracis

Elizabeth Bode  1 , William Hurtle, David Norwood
Affiliations

Real-time PCR assay for a unique chromosomal sequence of Bacillus anthracis

Elizabeth Bode et al. J Clin Microbiol. 2004 Dec.

Abstract

Real-time PCR has become an important method for the rapid identification of Bacillus anthracis since the 2001 anthrax mailings. Most real-time PCR assays for B. anthracis have been developed to detect virulence genes located on the pXO1 and pXO2 plasmids. In contrast, only two published chromosomal targets exist, the rpoB gene and the gyrA gene. In the present study, subtraction-hybridization with a plasmid-cured B. anthracis tester strain and a Bacillus cereus driver was used to find a unique chromosomal sequence. By targeting this region, a real-time assay was developed with the Ruggedized Advanced Pathogen Identification Device. Further testing has revealed that the assay has 100% sensitivity and 100% specificity, with a limit of detection of 50 fg of DNA. The results of a search for sequences with homology with the BLAST program demonstrated significant alignment to the recently published B. anthracis Ames strain, while an inquiry for protein sequence similarities indicated homology with an abhydrolase from B. anthracis strain A2012. The importance of this chromosomal assay will be to verify the presence of B. anthracis independently of plasmid occurrence.

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Figures

FIG. 1.
FIG. 1.
DNA products following nested PCR. Lanes 1 and 8, DNA PCR ladder (Gene Choice, Frederick, Md.); lane 2, subtracted B. anthracis dANR; lane 3, unsubtracted B. anthracis dANR; lane 4, subtracted E. coli; lane 5, unsubtracted E. coli; lane 6, subtracted control E. coli; lane 7, empty. The values at the sides of the gel are in base pairs.
FIG. 2.
FIG. 2.
Real-time PCR results for determination of efficacy. NTC, no template control; PGAMES, picograms of strain Ames; FGAMES, femtograms of strain Ames.
See this image and copyright information in PMC

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