Abstract
The revelation in May 2015 of the shipment of γ irradiation–inactivated wild-type Bacillus anthracis spore preparations containing a small number of live spores raised concern about the safety and security of these materials. The finding also raised doubts about the validity of the protocols and procedures used to prepare them. Such inactivated reference materials were used as positive controls in assays to detect suspected B. anthracis in samples because live agent cannot be shipped for use in field settings, in improvement of currently deployed detection methods or development of new methods, or for quality assurance and training activities. Hence, risk-mitigated B. anthracis strains are needed to fulfill these requirements. We constructed a genetically inactivated or attenuated strain containing relevant molecular assay targets and tested to compare assay performance using this strain to the historical data obtained using irradiation-inactivated virulent spores.
Highlights
We demonstrated an alternate approach that can potentially minimize risks associated with using BSATs and perhaps eliminate their use in some applications
To maintain a slightly higher copy number of the introduced plasmid assay targets, we introduced the assay targets into the ∆pXO1 backbone rather than into the chromosome in a strain lacking both pXO1 and pXO2
This way, assay results would be comparable in terms of copy numbers and cycle threshold values to historical assay data produced from a strain such as Ames
Summary
DNA concentration was determined using a NanoDrop (Thermo Fisher Scientific) We diluted extracts such that PCR reactions were performed starting with either 10 or 50 genomic copies. Comparison of Assay Performance of BAP708 Spores to Historical Data from Irradiation-Inactivated Select Agent B. anthracis Spores We prepared liquid and filter extracted samples according to established protocols using 2 separate aliquots of live and irradiation-inactivated BAP708 spore preparations. We diluted spore stock (≈2.0 × 1010 CFU/mL) in 1× phosphate-buffered saline to a spiking concentration of 2.0 × 106 CFU/mL and either extracted the stock directly as liquid samples or spiked it onto quarter filters, allowed to it dry, and extracted it as filter samples. The measurements were done in quadruplicate, and the minimal spore concentration that crossed the threshold was reported as the limit of detection using each spore preparation
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