Real-Time PCR Identification of Unique <i>Bacillus anthracis</i> Sequences

Cieślik, P.; Knap, J.; Kołodziej, M.; Mirski, T.; Joniec, J.; Graniak, G.; Żakowska, D.; Winnicka, I.; Bielawska-Drózd, Agata

doi:10.14712/fb2015061050178

Folia Biologica > Archive > 2015, Vol. 61 > Issue 5 > Real-Time PCR Identification of Unique…

Fol. Biol. 2015, 61, 178-183

https://doi.org/10.14712/fb2015061050178

Real-Time PCR Identification of Unique Bacillus anthracis Sequences

P. Cieślik¹, J. Knap², M. Kołodziej¹, T. Mirski¹, J. Joniec¹, G. Graniak¹, D. Żakowska¹, I. Winnicka³, Agata Bielawska-Drózd¹

¹Biological Threats Identification and Countermeasure Centre of the General Karol Kaczkowski Military Institute of Hygiene and Epidemiology, Puławy, Poland
²Warsaw Medical University, Second Faculty of Medicine, Department of Epidemiology, Warsaw, Poland
³General Karol Kaczkowski Military Institute of Hygiene and Epidemiology, Epidemiology Department, Warsaw, Poland

Received April 2015
Accepted August 2015

Bacillus anthracis is a spore-forming, Grampositive microorganism. It is a causative agent of anthrax, a highly infectious disease. It belongs to the “Bacillus cereus group”, which contains other closely related species, including Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus pseudomycoides. B. anthracis naturally occurs in soil environments. The BA5345 genetic marker was used for highly specific detection of B. anthracis with TaqMan probes. The detection limit of a real-time PCR assay was estimated at the level of 16.9 copies (CI_95% - 37.4 to 37.86, SD = 0.2; SE = 0.118). Oligonucleotides designed for the targeted sequences (within the tested locus) revealed 100 % homology to B. anthracis strain reference sequences deposited in the database (NCBI) and high specificity to all tested B. anthracis strains. Additional in silico analysis of plasmid markers pag and cap genes with B. anthracis strains included in the database was carried out. Our study clearly indicates that the BA5345 marker can be used with success as a chromosomal marker in routine identification of B. anthracis; moreover, detection of plasmid markers indicates virulence of the examined strains.