The detection and quantification of Bacillus thuringiensis spores from soil and swabs using quantitative PCR as a model system for routine diagnostics of Bacillus anthracis

• Published in: Journal of applied microbiology Vol. 123; no. 1; pp. 116 - 123
• Main Authors: Sedlackova, V., Dziedzinska, R., Babak, V., Kralik, P.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.07.2017
• Subjects: Bacillus anthracis; Bacillus thuringiensis; Deoxyribonucleic acid; DNA; Extraction; isolation; Microorganisms; Military; Pathogenicity; Pathogens; Plates; Polymerase chain reaction; qPCR; soil; Soils; spore; Spores; swab; Bacillus anthracis; swab; Bacillus thuringiensis; kit; qPCR; isolation; soil; spore
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/jam.13445

Aims To optimize the DNA isolation for the routine detection and quantification of bacillary spores in soil and swabs. The procedure is primarily intended for diagnostics of Bacillus anthracis spores, but due to its high pathogenicity, B. thuringiensis served as its surrogate organism. Methods and Results Various commercial kits for soils and swabs in combination with quantitative PCR were tested with different results. The PowerSoil DNA kit and the Ultra Clean Microbial DNA kit gave the best results for the extraction from soil and swabs, respectively. Extra beating led to considerably higher yields of DNA. The effectiveness of isolation reached 23% for DNA isolation from soil and 13% from swabs. The limit of detection was assessed to be 8·85 × 103 from 250 mg of soil and 2·79 × 103 from a swab inoculated with 100 μl of spore suspension. Conclusions The optimized protocol is suitable for direct isolation and quantification of bacillary spores without any previous culturing. Significance and Impact of the Study In contrast to previous studies, the isolation and quantification of spores was performed directly from the sample, without previous culture of spores on plates. Therefore, the method is suitable for such conditions where previous culturing is not possible, such as in military installations under field conditions.