Abstract
The anthrax pathogen Bacillus anthracis poses a significant threat to human health. Identification of B. anthracis is challenging because of the bacterium’s close genetic relationship to other Bacillus cereus group species. Thus, molecular detection is founded on species-specific PCR targeting single-copy genes. Here, we validated a previously recognized multi-copy target, a species-specific single nucleotide polymorphism (SNP) present in 2–5 copies in every B. anthracis genome analyzed. For this, a hydrolysis probe-based real-time PCR assay was developed and rigorously tested. The assay was specific as only B. anthracis DNA yielded positive results, was linear over 9 log10 units, and was sensitive with a limit of detection (LoD) of 2.9 copies/reaction. Though not exhibiting a lower LoD than established single-copy PCR targets (dhp61 or PL3), the higher copy number of the B. anthracis–specific 16S rRNA gene alleles afforded ≤2 unit lower threshold (Ct) values. To push the detection limit even further, the assay was adapted for reverse transcription PCR on 16S rRNA transcripts. This RT-PCR assay was also linear over 9 log10 units and was sensitive with an LoD of 6.3 copies/reaction. In a dilution series of experiments, the 16S RT-PCR assay achieved a thousand-fold higher sensitivity than the DNA-targeting assays. For molecular diagnostics, we recommend a real-time RT-PCR assay variant in which both DNA and RNA serve as templates (thus, no requirement for DNase treatment). This can at least provide results equaling the DNA-based implementation if no RNA is present but is superior even at the lowest residual rRNA concentrations.
Highlights
Within the genus Bacillus, the notorious anthrax pathogen Bacillus anthracis poses the greatest risk for humans, mammal livestock, and wildlife [1]
The 16S single nucleotide polymorphism (SNP) BA probe was verified in silico against the NCBI database to be highly specific for B. anthracis, i.e., all B. anthracis genomes showed a 100% match, and only genomes of a few other bacterial isolates exhibited identical sequences
E.g., a small number of Sphingomonas spp. Others, such as a few genomes annotated as Staphylococus aureus, had the same one-base-pair mismatch at the SNP-position and were identical to other B. cereus s.l. genomes, hybridizing perfectly against the alternative “16S SNP BC probe” (Figure S1)
Summary
Within the genus Bacillus, the notorious anthrax pathogen Bacillus anthracis poses the greatest risk for humans, mammal livestock, and wildlife [1]. Only obligatory pathogenic B. anthracis (and a few B. anthracis-like bacilli) features a unique suite of pathogenicity factors rendering the endospore-forming bacterium a first-rate biothreat agent These factors are encoded on two plasmids called pXO1 and pXO2. Plasmid pXO1 encodes the anthrax toxin genes producing the lethal toxin (gene products of pagA and lef ) and edema toxin (gene products of pagA and cya) [1]. These toxins damage host cells on various levels [3]. B. anthracis belongs to the very closely related Bacillus cereus sensu lato group. Besides the better-known species B. cereus sensu stricto, B. anthracis, or B. thuringiensis, the group comprises several other familiar species such as B. weihenstephanensis, B. mycoides, B. cytotoxicus, and a variety of lesser-characterized members [5]
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