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; 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.

Keywords: biothreat agents; isothermal amplification; quantitative PCR.

FIG 1

Main steps carried out in a laboratory tent in the field for sample processing from receipt to the final analyses. The laboratory tent was deployed in the field (A), and samples were brought to the laboratory by the Polish State Firemen CBRN sampling team (B). Samples were processed in a glove box to inactivate potential bioagents present in the sample (C and D), and nucleic acids were then extracted outside the glove box (E). qPCR and RPA assays were carried out, and data were analyzed (F).

FIG 2

RPA assay optimization. Optimization was achieved using the TwistAmp Basic amplification kit, as illustrated for the lef target gene. (A) Relative positions of RPA primer pairs (sets 1 to 3) used in screening and the part of the target where the lef probe was designed. (B) Results of gel electrophoresis analysis following target amplification with the indicated sets of primers. Samples 1 and 2 were NTC samples prepared in pre-PCR and post-PCR rooms, respectively, while sample 3 contained the target DNA.

FIG 3

RPA plots. Amplification was carried out with 10-fold serial dilutions of BA_5345 (A), lef (B), and capA (C) target sequences, as well as NTC samples.

FIG 4

Analytical sensitivity and quantitative ranges of BA_5345, lef, and capA RPA assays. (A) LOD values, as determined by probit regression analysis. (B) Assay linearity ranges.