Affiliation:
1. Centre de Technologies Moléculaires Appliquées, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Brussels, Belgium
2. Biothreats Unit, Defense Laboratories Department, Belgian Armed Forces, Brussels, Belgium
3. Institute of Veterinary Bacteriology, Vetsuisse, University of Bern, Bern, Switzerland
Abstract
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 10
2
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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
16 articles.
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