Sensitive and Specific Recombinase Polymerase Amplification Assays for Fast Screening, Detection, and Identification of Bacillus anthracis in a Field Setting
Authors: Bentahir Mostafa , Ambroise Jérôme , Delcorps Cathy , Pilo Paola , Gala Jean-Luc 
Published: April 2018

Four isothermal recombinase polymerase amplification (RPA) assays were developed for fast in-field identification ofBacillus anthracis. The RPA assays targeted three specific sequences (i.e., the BA_5345 chromosomal marker, the lethal factorlef[from pXO1], and the capsule-biosynthesis-relatedcapA[from pXO2]) and a conserved sequence in the adenylate cyclase gene (adk) for theBacillus cereusgroup.B. anthracis-specific RPA assays were tested first with purified genomic DNAs (n= 60), including 11 representatives ofB. anthracis, and then with soil (n= 8) and white powder (n= 8) samples spiked with inactivatedB. anthracisspores 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 20B. anthracisstrains). RPA assays displayed 100% specificity and sensitivity. The hands-off turnaround times at 42°C ranged from 5 to 6 min for 102genomic copies. The analytical sensitivity of each RPA assay was ∼10 molecules per reaction. In addition, the BA_5345 andadkRPA assays were assessed under field conditions with a series of surface swabs (n= 13, including 11 swabs contaminated withB. thuringiensisspores) that were blindly brought to the field laboratory by a chemical, biological, radiological, and nuclear (CBRN) sampling team. None of the 13 samples, except the control, tested positive forB. anthracis, and all samples that had been harvested from spore-contaminated surfaces tested positive with theadkRPA assay. All threeB. anthracis-specific RPA assays proved suitable for rapid and reliable identification ofB. anthracisand therefore could easily be used by first responders under field conditions to quickly discriminate between a deliberate release ofB. anthracisspores and a hoax attack involving white powder.

IMPORTANCEIn recent decades, particularly following the 11 September 2001 and Amerithrax attacks, the world has experienced attempts to sow panic and chaos in society through thousands of white-powder copycats using household powders to mimic real bioterrorism attacks. In such circumstances, field-deployable detection methods are particularly needed to screen samples collected from the scene. The aim is to test the samples directly using a fast and reliable assay for detection of the presence ofB. anthracis. While this would not preclude further confirmatory tests from being performed in reference laboratories, it would bring useful, timely, and relevant information to local crisis managers and help them make appropriate decisions without having to wait for quantitative PCR results (with turnaround times of a few hours) or phenotypic identification and sequencing (with turnaround times of a few days). In the current investigation, we developed a set of isothermal RPA assays for the rapid screening and identification ofB. anthracisin powders and soil samples, with the purpose of discriminating a deliberate release ofB. anthracisspores from a hoax attack involving white powder; this would also apply to dispersion by spraying of aerosolized forms ofB. anthracis. Further work is now ongoing to confirm the first observations and validate the on-site use of these assays by first responders.