Evaluation of a Highly Efficient DNA Extraction Method for Bacillus anthracis Endospores

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.

Enzyme-Linked Phage Receptor Binding Protein Assays (ELPRA) Enable Identification of Bacillus anthracis Colonies

“…A bacterial colony grown on blood agar was chemically inactivated and DNA isolated using the MasterPure™ Gram Positive DNA Purification kit (Lucigen, Middleton, WI, USA) as described for Gram-positive bacteria, with minor modifications as described in [ 19 ]. DNA concentrations were quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Dreieich, Germany), according to the supplier’s protocol.…”

Reevaluating limits of detection of 12 lateral flow immunoassays for the detection of Yersinia pestis , Francisella tularensis , and Bacillus anthracis spores using viable risk group‐3 strains

“…For B. anthracis, previous studies have demonstrated that a 100% efficient DNA isolation from the purified spores is not achievable (Knüpfer et al . 2020). Thus, for quantifying the target cells used for LOD‐determination, we relied on the ddPCR results for Y. pestis and F. tularensis and microscopic counting results for B. anthracis .…”

“…Since quantitative DNA extraction from B. anthracis spores is very difficult even using optimized DNA isolation procedures (Knüpfer et al . 2020), spore concentration was quantified by microscopic counting and by plating on LB‐Agar. For each organism, quantified samples were serially diluted in 1 : 10 steps before application to LFA, with Y. pestis and F. tularensis diluted in a 0·9% NaCl solution, and B. anthracis spores diluted in sterile water.…”

Development and Validation of Ribosomal RNA-Targeted Reverse Transcription Real-Time PCR Assays for the Sensitive and Rapid Diagnostics of High Consequence Pathogens

“…Accordingly, the only hitherto existing rtPCR targeting the 16S rRNA gene of Y. pestis failed to discriminate between Y. pestis and other bacteria of the genus Yersinia (Tomaso et al, 2003). For F. tularensis, a rtPCR successfully targeting the 16S rDNA has been described (Knüpfer et al, 2020). However, in its present form, the sensitivity of this assay is about 86 colony forming units (CFU)/ml (Knüpfer et al, 2020) and thus not optimal.…”

“…For F. tularensis, a rtPCR successfully targeting the 16S rDNA has been described (Knüpfer et al, 2020). However, in its present form, the sensitivity of this assay is about 86 colony forming units (CFU)/ml (Knüpfer et al, 2020) and thus not optimal.…”