In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

J. Agren, R.A. Hamidjaja, T. Hansen, R.C. Ruuls, S. Thierry, H. Vigre, I. Janse, A. Sundström, B. Segerman, M.G.J. Koene, Ch. Löfström, B. van Rotterdam, S. Derzelle

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.
Original languageEnglish
Pages (from-to)671-685
JournalVirulence
Volume4
Issue number8
DOIs
Publication statusPublished - 2013

Fingerprint

Bacillus anthracis
Computer Simulation
Polymerase Chain Reaction
Prophages
Bacillus
Anthrax
Animal Diseases
Cross Reactions
Zoonoses
Genetic Markers
Virulence
Limit of Detection
In Vitro Techniques
Plasmids
Chromosomes
Genome
DNA
Genes

Keywords

  • real-time pcr
  • single-nucleotide polymorphisms
  • closely-related bacteria
  • rapid-detection methods
  • cereus group
  • environmental-samples
  • multiplex pcr
  • toxin genes
  • nasal swabs
  • identification

Cite this

Agren, J., Hamidjaja, R. A., Hansen, T., Ruuls, R. C., Thierry, S., Vigre, H., ... Derzelle, S. (2013). In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences. Virulence, 4(8), 671-685. https://doi.org/10.4161/viru.26288
Agren, J. ; Hamidjaja, R.A. ; Hansen, T. ; Ruuls, R.C. ; Thierry, S. ; Vigre, H. ; Janse, I. ; Sundström, A. ; Segerman, B. ; Koene, M.G.J. ; Löfström, Ch. ; van Rotterdam, B. ; Derzelle, S. / In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences. In: Virulence. 2013 ; Vol. 4, No. 8. pp. 671-685.
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abstract = "Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100{\%} specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.",
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Agren, J, Hamidjaja, RA, Hansen, T, Ruuls, RC, Thierry, S, Vigre, H, Janse, I, Sundström, A, Segerman, B, Koene, MGJ, Löfström, C, van Rotterdam, B & Derzelle, S 2013, 'In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences', Virulence, vol. 4, no. 8, pp. 671-685. https://doi.org/10.4161/viru.26288

In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences. / Agren, J.; Hamidjaja, R.A.; Hansen, T.; Ruuls, R.C.; Thierry, S.; Vigre, H.; Janse, I.; Sundström, A.; Segerman, B.; Koene, M.G.J.; Löfström, Ch.; van Rotterdam, B.; Derzelle, S.

In: Virulence, Vol. 4, No. 8, 2013, p. 671-685.

Research output: Contribution to journalArticleAcademicpeer-review

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T1 - In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

AU - Agren, J.

AU - Hamidjaja, R.A.

AU - Hansen, T.

AU - Ruuls, R.C.

AU - Thierry, S.

AU - Vigre, H.

AU - Janse, I.

AU - Sundström, A.

AU - Segerman, B.

AU - Koene, M.G.J.

AU - Löfström, Ch.

AU - van Rotterdam, B.

AU - Derzelle, S.

PY - 2013

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N2 - Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.

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KW - real-time pcr

KW - single-nucleotide polymorphisms

KW - closely-related bacteria

KW - rapid-detection methods

KW - cereus group

KW - environmental-samples

KW - multiplex pcr

KW - toxin genes

KW - nasal swabs

KW - identification

U2 - 10.4161/viru.26288

DO - 10.4161/viru.26288

M3 - Article

VL - 4

SP - 671

EP - 685

JO - Virulence

JF - Virulence

SN - 2150-5594

IS - 8

ER -