Abstract
ABSTRACTFour isothermal recombinase polymerase amplification (RPA) assays were developed for fast in-field identification of Bacillus anthracis. The RPA assays targeted three specific sequences (i.e., the BA_5345 chromosomal marker, the lethal factor lef [from pXO1], and the capsule-biosynthesis-related capA [from pXO2]) and a conserved sequence in the adenylate cyclase gene (adk) for the Bacillus cereus group. B. anthracis-specific RPA assays were tested first with purified genomic DNAs (n = 60), including 11 representatives of B. anthracis, and then with soil (n = 8) and white powder (n = 8) samples spiked with inactivated B. anthracis 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 B. anthracis strains). RPA assays displayed 100% specificity and sensitivity. The hands-off turnaround times at 42°C ranged from 5 to 6 min for 102 genomic copies. The analytical sensitivity of each RPA assay was ∼10 molecules per reaction. In addition, the BA_5345 and adk RPA assays were assessed under field conditions with a series of surface swabs (n = 13, including 11 swabs contaminated with B. thuringiensis spores) 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 for B. anthracis, and all samples that had been harvested from spore-contaminated surfaces tested positive with the adk RPA assay. All three B. anthracis-specific RPA assays proved suitable for rapid and reliable identification of B. anthracis and therefore could easily be used by first responders under field conditions to quickly discriminate between a deliberate release of B. anthracis spores and a hoax attack involving white powder.IMPORTANCE In 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 of B. 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 of B. anthracis in powders and soil samples, with the purpose of discriminating a deliberate release of B. anthracis spores from a hoax attack involving white powder; this would also apply to dispersion by spraying of aerosolized forms of B. anthracis. Further work is now ongoing to confirm the first observations and validate the on-site use of these assays by first responders.
Highlights
Four isothermal recombinase polymerase amplification (RPA) assays were developed for fast in-field identification of Bacillus anthracis
B. anthracis spores, as well as the need for assays based on amplification of sequences from the pXO1 and pXO2 virulence plasmids combined with specific chromosomal markers [9, 10]
It is noteworthy that a panel of 10 RPA assays was previously tested for detection of biothreat agents, including B. anthracis [19]
Summary
Four isothermal recombinase polymerase amplification (RPA) assays were developed for fast in-field identification of Bacillus anthracis. Following the 11 September 2001 and Amerithrax attacks, the world has experienced several attempts to sow panic and chaos in society through deliberate dispersion by thousands of white-powder copycats using household powders to mimic real bioterrorism attacks [5,6,7] Under such circumstances, rapid and reliable detection and identification methods are needed to quickly screen samples collected from the scene, i.e., to test directly for the presence of B. anthracis. Isothermal amplification enables health care workers in remote locations to quickly analyze samples for the presence of nucleic acids from a range of infectious agents, using field-deployable assays [11,12,13,14,15] Among these methods, loopmediated isothermal amplification (LAMP) [16] and recombinase polymerase amplification (RPA) [17] assays have become increasingly popular, and they are proposed in the setting of bioterrorism for rapid detection of biothreats, including B. anthracis [18,19,20]. Only a few, e.g., purA and pclR single-nucleotide polymorphisms (SNPs), as well as BA_5345, PL3, and BA5357 genes, were found to be unique to B. anthracis [9, 25]
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