| 1. |
- Thierry, S., et al.
(författare)
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A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis
- 2013
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Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 95:3, s. 357-365
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Tidskriftsartikel (refereegranskat)abstract
- Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2. ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis. © 2013 Elsevier B.V.
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| 2. |
- Ågren, Joakim, et al.
(författare)
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In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences
- 2013
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Ingår i: Virulence. - : Taylor and Francis Inc.. - 2150-5594 .- 2150-5608. ; 4:8
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Tidskriftsartikel (refereegranskat)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. © 2013 Landes Bioscience.
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| 3. |
- Woudstra, C., et al.
(författare)
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Validation of a real-time PCR based method for detection of clostridium botulinum types C, D and their mosaic variants C-D and D-C in a multicenter collaborative trial
- 2013
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Ingår i: Anaerobe. - : Elsevier BV. - 1075-9964 .- 1095-8274. ; 22, s. 31-37
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Tidskriftsartikel (refereegranskat)abstract
- Two real-time PCR arrays based on the GeneDisc® cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated with avian and mammalian botulism. The GeneDisc® arrays developed as part of the DG Home funded European project 'AnibioThreat' were highly sensitive and specific when tested on pure isolates and naturally contaminated samples (mostly clinical specimen from avian origin). Results of the multicenter collaborative trial involving eight laboratories in five European Countries (two laboratories in France, Italy and The Netherlands, one laboratory in Denmark and Sweden), using DNA extracts issued from 33 pure isolates and 48 naturally contaminated samples associated with animal botulism cases, demonstrated the robustness of these tests. Results showed a concordance among the eight laboratories of 99.4%-100% for both arrays. The reproducibility of the tests was high with a relative standard deviation ranging from 1.1% to 7.1%. Considering the high level of agreement achieved between the laboratories these PCR arrays constitute robust and suitable tools for rapid detection of C.botulinum types C, D and mosaic types C-D and D-C. These are the first tests for C.botulinum C and D that have been evaluated in a European multicenter collaborative trial. © 2013 Elsevier Ltd.
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