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In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences
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2013 (English)In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 4, no 8Article in journal (Refereed) Published
Abstract [en]

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. © 2013 Landes Bioscience.

Place, publisher, year, edition, pages
Taylor and Francis Inc. , 2013. Vol. 4, no 8
Keywords [en]
Bacillus anthracis, Chromosomal marker, Detection, Diagnostic sensitivity, In silico analysis, Inter-laboratory trial, qPCR, Specificity, anthrax, Bacillus cereus, bacterial strain, chromosome banding pattern, computer model, controlled study, cross reaction, DNA extraction, fluorescence resonance energy transfer, fluorometry, limit of detection, nonhuman, point mutation, real time polymerase chain reaction, review, sensitivity and specificity, single nucleotide polymorphism, world health organization, animal, article, Bacillus thuringiensis, bacterial chromosome, biology, chromosome marker, comparative study, evaluation study, genetics, human, isolation and purification, methodology, molecular diagnosis, polymerase chain reaction, Animalia, Bacillus cereus group, bacterial DNA, primer DNA, Animals, Chromosomes, Bacterial, Computational Biology, DNA Primers, DNA, Bacterial, Humans, Molecular Diagnostic Techniques
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Natural Sciences
Identifiers
URN: urn:nbn:se:ri:diva-39017DOI: 10.4161/viru.26288Scopus ID: 2-s2.0-85052275253OAI: oai:DiVA.org:ri-39017DiVA, id: diva2:1324810
Note

 Funding details: Ministry of Home Affairs - Singapore, Ministry of Home Affairs - Singapore; Funding details: European Commission, European Commission; Funding details: European Geosciences Union, European Geosciences Union; Funding details: Myndigheten för Samhällsskydd och Beredskap, Swedish Civil Contingencies Agency; Funding text 1: Pia Engelsmann, DTU, is acknowledged for excellent technical assistance. This research was supported by/executed in the framework of the EU-project AniBioThreat (Grant Agreement: Home/2009/ISEC/AG/191) with the financial support from the Prevention of and Fight against Crime Programme of the European Union, European Commission—Directorate General Home Affairs. This publication reflects the views only of the authors, and the European Commission cannot be held responsible for any use that may be made of the information contained therein. This work was also supported by the Swedish Civil Contingencies Agency (MSB).

Available from: 2019-06-14 Created: 2019-06-14 Last updated: 2019-06-14Bibliographically approved

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