| Summary: | <b>: </b>Prompt and accurate detection of <i>Bacillus anthracis </i>spores is crucial in the event of intentional spore dissemination in order to reduce the number of expected casualties. Specific identification of these spores from environmental samples is both challenging and time-consuming. This is due to the high homology with other Bacillus species as well as the complex composition of environmental samples, which further impedes assay sensitivity. Previously, we showed that a short incubation of <i>B.anthracis</i> spores in a defined growth medium results in rapid germination, bacterial growth, and secretion of toxins, including protective antigen. In this work, we tested whether coupling the incubation process to a newly developed immune-assay will enable the detection of secreted toxins as markers for the presence of spores in environmental samples. The new immune assay is a flow cytometry-based multiplex that simultaneously detects a protective antigen, lethal factor, and edema factor. Our combined assay detects 1 × 10<sup>3</sup>−1 × 10<sup>4</sup>/mL spores after a 2 h incubation followed by the ~80 min immune-multiplex detection. Extending the incubation step to 5 h increased assay sensitivity to 1 × 10<sup>2</sup>/mL spore. The protocol was validated in various environmental samples using attenuated or fully virulent <i>B. anthracis</i> spores. There was no substantial influence of contaminants derived from real environmental samples on the performance of the assay compared to clean samples, which allow the unequivocal detection of 3 × 10<sup>3</sup>/mL and 3 × 10<sup>2</sup>/mL spores following 2 and 5 hour’s incubation, respectively. Overall, we propose this method as a rapid, sensitive, and specific procedure for the identification of <i>B. anthracis</i> spores in environmental samples.
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