Sensitive and Specific Recombinase Polymerase Amplification Assays for Fast Screening, Detection, and Identification of Bacillus anthracis in a Field Setting

Abstract

Four 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; th...

Evaluation of real-time PCR based on SYBR Green I fluorescent dye for detection of Bacillus anthracis strains in biological samples

“…For the detection of the cap gene, Bentahir et al . (2) achieved a regression coefficient higher than 0.995 for pure cultures. In our studies, the high regression coefficient attained in SYBR Green I real-time PCRs with the use of the rpoB , pag , and capC genes indicates a high degree of reaction linearity.…”

Calibration of an Upconverting Phosphor-Based Quantitative Immunochromatographic Assay for Detecting Yersinia pestis, Brucella spp., and Bacillus anthracis Spores

“…Furthermore, the sample volume for the UPT-LF assay is 100 µL, in other words, only 10 3 bacterial cells are applied to the strip when the concentration of the bacterial sample is 10 4 cells mL −1 . For comparison, the sensitivities of real-time or isothermal recombinase PCR are about 10 copies per reaction for Y. pestis (Qu et al, 2010), B. anthracis (Antwerpen et al, 2008;Bentahir et al, 2018), and Brucella spp. (Zeybek et al, 2019), which is equivalent to 2 × 10 3 -1 × 10 4 cells mL −1 in a 1-5 µL sample volume of template for each PCR, thereby enrichment of the target bacteria by culturing or extraction of nucleic acid is often required (Kane et al, 2019).…”

Detection and Identification of Bacillus anthracis: From Conventional to Molecular Microbiology Methods

“…A pair of primers is used for the reaction and the amplification occurs thanks to the action of three proteins: a strand-displacing polymerase, a recombinase, and a single-stranded DNA binding protein. The reaction is conducted for about 30 min at 38 • C. Euler et al [58] designed RPA primers for pagA (pXO1) and capC (pXO2), whereas Bentahir et al [59] designed RPA primers for BA_5345 (the chromosomal marker), lef (pXO1), and capA (pXO2). High specificity of the results was shown.…”