Affiliation:
1. Department of Microbiology, National Veterinary Research Institute , 24-100 Pulawy , Poland
Abstract
Abstract
Introduction: The aim of the study was the application and evaluation of real-time PCRs based on the fluorescence of SYBR Green I intercalating dye for the detection of three Bacillus anthracis genes in contaminated liver and blood samples. The goals for detection were rpoB gene as a chromosomal marker, pag gene located on plasmid pXO1, and capC gene located on plasmid pXO2.
Material and Methods: Five B. anthracis strains were used for the experiments. Additionally, single strains of other species of the genus Bacillus, i.e. B. cereus, B. brevis, B. subtilis, and B. megaterium, and strains of six other species were used for evaluation of the specificity of the tests. Three SYBR Green I real-time PCRs were conducted allowing confirmation of B. anthracis in the biological samples.
Results: The observation of amplification curves in real-time PCRs enabled the detection of the chromosomally encoded rpoB gene, pag gene, and capC gene of B. anthracis. The specificity of the tests was confirmed by estimation of the melting temperature of the PCR products. The sensitivity and linearity of the reactions were determined using regression coefficients. Strains of other microbial species did not reveal real-time PCR products.
Conclusion: All real-time PCRs for the detection of B. anthracis in biological samples demonstrated a significant sensitivity and high specificity.
Reference30 articles.
1. 1. Adone R., Pasquali P., La Rosa G., Marianelli C., Muscillo M., Fasanella A., Francia M., Ciuchini F.: Sequence analysis of the genes encoding for the major virulence factors of Bacillus anthracis vaccine strain Carbosap. J Appl Microbiol 2002, 93, 117–121.
2. 2. Bentahir M., Ambroise J., Delcorps C., Pilo P., Gala J.L.: Sensitive and specific recombinase polymerase amplification for fast screening, detection, and identification of Bacillus anthracis in a field setting. Appl Environ Microbiol 2018, 84, 1–13.
3. 3. Beyer W., Turnbull P.C.B.: Anthrax in animals. Mol Aspects Med 2009, 30, 481–489.
4. 4. Bode E., Hurtle W., Norwood D.: Real-time PCR assay for a unique chromosomal sequence of Bacillus anthracis. J Clin Microbiol 2004, 42, 5825–5831.
5. 5. Budniak S., Kędrak-Jabłonska A., Reksa M., Szczawińska A., Borkowska-Opacka B.: Application of the real-time PCR method with the intercalating dye SYBR Green I for the detection of genes located on plasmids pXO1 and pXO2 as well as the specific chromosomal rpoB sequence of Bacillus anthracis strains. Med Weter 2010, 66, 630–634.
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