Medycyna Wet. 66 (9), 630-634, 2010
| Budniak S., Kędrak-Jabłońska 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 |
| The aim of the study was to apply the real-time PCR method with the intercalating dye SYBR Green I for the detection of genes of Bacillus anthracis located on plasmids pXO1 and pXO2 and the specific chromosomal rpoB sequence. The research was conducted on one vaccinal and four field strains of Bacillus anthracis. The assessment of the specificity of the tests involved isolates of other species of the genus Bacillus as well as strains of six other species of microorganisms. The studies were conducted with the PCR QuantiTect kit (Qiagen) and primers specific for the gene pag coding PA protein, gene cap coding capsule, and primers for the amplification of the specific chromosomal sequence. PCR enabled the detection of all genes under examination by the observation of amplification curves. The specificity of real-time PCR was confirmed by estimating melting temperatures of PCR products. It was shown that the melting temperatures of amplicons obtained in the reaction were 78ºC, 79°C and 76°C in cases of detecting the chromosomal rpoB sequence, pag gene, and cap-C gene, respectively. The sensitivity and linearity of the reactions were determined using a regression coefficient. A high regression coefficient of 0.99 was demonstrated for all the reactions. The real-time tests were highly sensitive and specific. |
| Keywords: Bacillus anthracis, real-time PCR, SYBR Green I |