Abstract
A variety of methods have been established in order to optimize the accessibility of DNA originating from Bacillus anthracis cells and endospores to facilitate highly sensitive molecular diagnostics. However, most endospore lysis techniques have not been evaluated in respect to their quantitative proficiencies. Here, we started by systematically assessing the efficiencies of 20 DNA extraction kits for vegetative B. anthracis cells. Of these, the Epicentre MasterPure kit gave the best DNA yields and quality suitable for further genomic analysis. Yet, none of the kits tested were able to extract reasonable quantities of DNA from cores of the endospores. Thus, we developed a mechanical endospore lysis protocol, facilitating the extraction of high-quality DNA. Transmission electron microscopy or the labelling of spores with the indicator dye propidium monoazide was utilized to assess lysis efficiency. Finally, the yield and quality of genomic spore DNA were quantified by PCR and they were found to be dependent on lysis matrix composition, instrumental parameters, and the method used for subsequent DNA purification. Our final standardized lysis and DNA extraction protocol allows for the quantitative detection of low levels (<50 CFU/mL) of B. anthracis endospores and it is suitable for direct quantification, even under resource-limited field conditions, where culturing is not an option.
Highlights
We showed that DNA yield, purity, and integrity were the highest for the test organisms B. anthracis and F. tularensis when using the MasterPure Complete DNA and RNA Purification kit supplemented with RNAse, glycogen, and tracing-dye Roti®PinkDNA
Despite the large number of methods, assays, and instrumentation used for the detection of B. anthracis, classical bacteriology, e.g., cultivation and microscopy, remains a standard for diagnosis, even though this requires at least 24 h to complete
QPCR has been widely used for the detection of pathogens in clinical and environmental samples and in a large number of laboratories and institutions, DNA is usually extracted with the QIAamp DNA Mini kit or the QIAamp Blood and Tissue kit [42]
Summary
The aim of this study was to test 20 commercially available kits for the extraction of genomic DNA from vegetative cells and spores of B. anthracis.
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