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Rapid and Sensitive Multiplex Assay for the Detection of B. anthracis Spores from Environmental Samples.

ABSTRACT: : Prompt and accurate detection of Bacillus anthracis 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 B.anthracis 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 × 103-1 × 104/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 × 102/mL spore. The protocol was validated in various environmental samples using attenuated or fully virulent B. anthracis 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 × 103/mL and 3 × 102/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 B. anthracis spores in environmental samples.

SUBMITTER: Makdasi E

PROVIDER: S-EPMC7157734 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Rapid and Sensitive Multiplex Assay for the Detection of B. anthracis Spores from Environmental Samples.

Makdasi Efi E Laskar Orly O Glinert Itai I Alcalay Ron R Mechaly Adva A Levy Haim H

Pathogens (Basel, Switzerland) 20200227 3

: Prompt and accurate detection of Bacillus anthracis 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 incubat ...[more]

PMID: 32120986

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Project description:BACKGROUND:Yersinia pestis is a contributing agent to the epidemic disease, plague, which killed an estimated 200 million people during historical times. In this study, a rapid, cheap, sensitive, and specific technique, the lateral flow assay (F1 strips), has been successfully developed to detect this pathogen, by using paired monoclonal antibodies (MAbs) against Y. pestis capsule like fraction 1 (F1) protein. Compared with the polyclonal antibody (PAb) based F1 strips, the Mab-based F1 strips have a remarkable increased detection limitation (10 to 100 folds). Furthermore, besides the limitation and specificity evaluation, the application of this F1 strip on simulated clinical samples indicate the LFA can be a good candidate to detect plague. METHODS:Recombinant F1 antigen was expressed and purified from a series of works. The various anti-F1 monoclonal antibodies generated from hybridoma cells were screened with the ELISA technique. To evaluate the feasibility of this Y. pestis F1 test strip, the F1 protein/Y. pestis was spiked into simulated clinical samples such as human serum, mouse bronchoalveolar lavage fluids, and mouse blood to mimic natural infection status. Additionally, this technique was applied to detect the Y. pestis in the environment-captured rats, to evaluate the practical usefulness of the strips. RESULTS:By using this MAb-based-LFA technique, 4 ng/ml of recombinant F1-protein and 103 CFU/ml of Y. pestis could be detected in less than 10 mins, which is at least 10-folds than that of the PAb format. On the other hand, although various Yersinia strains were applied to the strips, only Y. pestis strain showed a positive result; all other Yersinia species did not produce a positive signal, indicating the high efficiency and specificity of the MAb-based F1-strips. CONCLUSION:Based on our findings, we suggest that the MAb-format-LFA will be valuable as a diagnostic tool for the detection of Y. pestis. This report shows that the F1 strip is sufficient to support not only the detection of plague in simulated clinical samples, but also it may be a good candidate to meet the epidemiological surveillance during an outbreak of the biological warfare.

Dataset Information

Rapid and Sensitive Multiplex Assay for the Detection of B. anthracis Spores from Environmental Samples.

Publications

Rapid and Sensitive Multiplex Assay for the Detection of <i>B. anthracis</i> Spores from Environmental Samples.

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