Real-Time PCR Detection of Bacillus anthracis by Lambda_Ba03 Prophage Genes

The aim of the study was to develop a set of primers and fluorescent probes for the detection of two chromosomal targets of Bacillus anthracis using real-time PCR based on the lambda_Ba03 prophage genes.

Materials and methods. BLAST analysis of B. anthracis chromosomal DNA identified two target genes in the region of lambdaBa03 prophage, BA_5358 (AE016879.1: 4852332..4853642) and BA_5361 (AE016879.1: 4855298..4856278). The designed primers and fluorescent hydrolysable TaqMan probes for simultaneous detection of B. anthracis chromosomal DNA by two stated genes were tested in qPCR for sensitivity and specificity.

Results and discussion. Studies performed on chromosomal DNA samples of closely related bacteria (B. cereus, B. thuringiensis, B. subtilis, B. clausii) have shown 100 % specificity of the developed sets of primers/probes. The sensitivity of the devised multiplex kit, tested on DNA samples of the m55-VNIIVViM vaccine strain and archival DNA samples of B. anthracis, reached 100 fg of bacterial DNA, which sets the limit of sensitivity at 17 genomes per reaction. The developed multiplex kit can be used as a separate tool for research laboratories studying anthrax.

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