Sensitive and specific recombinase polymerase amplification set of assays for fast screening, detection and identification of Bacillus anthracis in a field setting.
(2018) Applied and Environmental Microbiology — (2018)
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- Four isothermal recombinase polymerase amplification assays (RPA) were developed for fast in-field identification of RPAs targeted three specific sequences (i.e. BA_5345 chromosomal marker, lethal factor -pXO1, and capsule biosynthesis -pXO2), and a conserved sequence of the group in the adenylate cyclase gene (). -specific RPA assays were first tested on purified genomic DNAs (n=60) containing 11 representatives of , then on soil (n=8) and white powder (n=8) samples spiked with inactivated spores and/or other biological agents. The RPA assays were also tested in another laboratory facility which blindly provided DNA and lysate samples (n=30, including 20 strains). RPA tests displayed 100% specificity and sensitivity. The hands-off turnaround time at 42°C ranged from 5 to 6 min for 10 genomic copies. The analytical sensitivity of each RPA was ∼10 molecules per reaction. Besides, BA_5345 and RPA assays were assessed in field conditions on a series of surface swabs (n=13 with 11 swabs contaminated with spores) and blindly dispatched to the field laboratory by a CBRN sampling team. None of the 13 samples, expect the control, tested positive for and all samples harvested on spores-contaminated surfaces tested positive with -RPA assay. All three -specific RPA assays proved suitable for rapid and reliable identification of and could therefore easily be used by first responders in field conditions to quickly discriminate between a deliberate release of spores and a hoax attack involving "white-powders". In recent decades, particularly following 9/11 and the Amerithrax attack, the world experienced several attempts to sow panic and chaos in the society through thousands of white-powder copycats using household powders to mimic a real bioterrorism attack. In such circumstances, field deployable detection methods are particularly needed to screen samples collected from the scene. The aim is to test them directly using a fast and reliable assay for detecting the presence of While this does not prevent further confirmatory tests to be performed in reference laboratories, it would bring useful, timely relevant information to the local crisis managers, and help them to take appropriate decisions without having to wait for qPCR results (turnaround time: few hours) or phenotypic identification and sequencing (turnaround time: few days). In the current investigation, we developed a set of isothermal RPA assays for a rapid screening and identification of in powders and soil samples with the purpose to discriminate a deliberate release of spores from a hoax attack involving "white-powders". This would also apply to dispersion by spraying aerosolized forms of Further work is now ongoing to confirm the first observations and validate the on-site use of these assays by first responders.
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Bentahir, M., Ambroise, J., Delcorps, C., Pilo, P., & Gala, J.-L. (2018). Sensitive and specific recombinase polymerase amplification set of assays for fast screening, detection and identification of Bacillus anthracis in a field setting. Applied and Environmental Microbiology. Submitted. https://doi.org/10.1128/AEM.00506-18 (Original work published 2018)