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

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 det...

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Autores principales:	Peter Braun, Martin Duy-Thanh Nguyen, Mathias C. Walter, Gregor Grass
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	anthrax <i>Bacillus anthracis</i> 16S rRNA detection identification real-time PCR Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/cb5cdc7771c349dcb7deff9c16c81cd6
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spelling	oai:doaj.org-article:cb5cdc7771c349dcb7deff9c16c81cd62021-11-25T17:54:28ZUltrasensitive Detection of <i>Bacillus anthracis</i> by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts10.3390/ijms2222122241422-00671661-6596https://doaj.org/article/cb5cdc7771c349dcb7deff9c16c81cd62021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/22/12224https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067The anthrax pathogen <i>Bacillus anthracis</i> poses a significant threat to human health. Identification of <i>B. anthracis</i> is challenging because of the bacterium’s close genetic relationship to other <i>Bacillus cereus</i> 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 <i>B. anthracis</i> genome analyzed. For this, a hydrolysis probe-based real-time PCR assay was developed and rigorously tested. The assay was specific as only <i>B. anthracis</i> DNA yielded positive results, was linear over 9 log<sub>10</sub> 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 (<i>dhp61</i> or <i>PL3</i>), the higher copy number of the <i>B. anthracis</i>–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 log<sub>10</sub> 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.Peter BraunMartin Duy-Thanh NguyenMathias C. WalterGregor GrassMDPI AGarticleanthrax<i>Bacillus anthracis</i>16S rRNAdetectionidentificationreal-time PCRBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12224, p 12224 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	anthrax <i>Bacillus anthracis</i> 16S rRNA detection identification real-time PCR Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	anthrax <i>Bacillus anthracis</i> 16S rRNA detection identification real-time PCR Biology (General) QH301-705.5 Chemistry QD1-999 Peter Braun Martin Duy-Thanh Nguyen Mathias C. Walter Gregor Grass Ultrasensitive Detection of <i>Bacillus anthracis</i> by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts
description	The anthrax pathogen <i>Bacillus anthracis</i> poses a significant threat to human health. Identification of <i>B. anthracis</i> is challenging because of the bacterium’s close genetic relationship to other <i>Bacillus cereus</i> 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 <i>B. anthracis</i> genome analyzed. For this, a hydrolysis probe-based real-time PCR assay was developed and rigorously tested. The assay was specific as only <i>B. anthracis</i> DNA yielded positive results, was linear over 9 log<sub>10</sub> 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 (<i>dhp61</i> or <i>PL3</i>), the higher copy number of the <i>B. anthracis</i>–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 log<sub>10</sub> 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.
format	article
author	Peter Braun Martin Duy-Thanh Nguyen Mathias C. Walter Gregor Grass
author_facet	Peter Braun Martin Duy-Thanh Nguyen Mathias C. Walter Gregor Grass
author_sort	Peter Braun
title	Ultrasensitive Detection of <i>Bacillus anthracis</i> by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts
title_short	Ultrasensitive Detection of <i>Bacillus anthracis</i> by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts
title_full	Ultrasensitive Detection of <i>Bacillus anthracis</i> by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts
title_fullStr	Ultrasensitive Detection of <i>Bacillus anthracis</i> by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts
title_full_unstemmed	Ultrasensitive Detection of <i>Bacillus anthracis</i> by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts
title_sort	ultrasensitive detection of <i>bacillus anthracis</i> by real-time pcr targeting a polymorphism in multi-copy 16s rrna genes and their transcripts
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/cb5cdc7771c349dcb7deff9c16c81cd6
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Ultrasensitive Detection of <i>Bacillus anthracis</i> by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts

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