Ultrasensitive Detection of Bacillus anthracis by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts

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.

Single-tube isothermal label-free fluorescent sensor for pathogen detection based on genetic signatures

“…The limit of detection (LOD) calculated using the 3σ-rule and 0–25 nM linear dynamic range is 1.7 nM and 0.6 nM for the 30-min and 60-min assay, respectively ( Figure 3B ). These LOD values, while unable to compete with the LODs of PCR-based assays ( Braun et al, 2021 ), are in the same range as the LOD reported for the molecular beacon probe (MBP) ( Kolpashchikov 2012 ). Unlike MBP, which cannot efficiently interrogate a structured nucleic acid target ( Nguyen et al, 2011 ; Reed et al, 2020 ), the split junction probe used in the aptamer-expressing assay can tolerate stable secondary/tertiary structures a natural RNA target generally acquires.…”

Identification of Universally Applicable and Species-Specific Marker Peptides for Bacillus anthracis

“…Despite these high similarities, we were able to identify eleven species-specific candidate marker peptides for BA, which allow for differentiating BA from all other species, including the closely related Bacillus cereus groups (see Table 1 ). These eleven markers are all chromosomally encoded and tie in with recent studies reporting the interest of researchers in chromosome-encoded genes, which would be preferable for the specific detection of BA, due to occasionally observed losses of virulence plasmids within environmental species and the occurrence of virulence plasmids in atypical Bacillus cereus strains [ 6 , 8 , 38 , 39 ].…”

“…As discussed previously, while methods targeting the virulence plasmids only verify the presence of the plasmids, species-specific molecular identification of BA can be achieved by using chromosomal targets [ 39 ]. In this context, it is especially interesting that the genes coding for the here identified potential marker candidates are chromosomally localized.…”

Reoccurring Bovine Anthrax in Germany on the Same Pasture after 12 Years

“…Independent of initial diagnostic PCR analysis performed by state health authorities, blood taken from the left nostril of the carcass ( Fig. 1A ) was inactivated and subjected to recently developed ultrasensitive 16S rRNA SNP (RT-)PCR ( 19 ) and phage RBP reporter-based rapid detection assays ( 18 ). Results confirmed the previous PCR tests, as phage RBP λ03Δ1–120 reporter-based ELPRA gave positive results when inactivated blood samples from the carcass were tested ( Fig.…”

“…For direct PCR-based detection of B. anthracis in blood samples, 100 μL inactivated blood sample was incubated at 95°C for 10 min to lyse cells and centrifuged at 5,000 × g for 2 min. Aliquots of 5 μL of the supernatant were then used as templates for 16S rRNA single nucleotide polymorphism (SNP) PCR or 16S rRNA SNP reverse transcription-PCR (RT-PCR) performed as described in reference 19 . Alternatively, total nucleic acid extractions of blood samples were used as templates.…”