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A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis
DTU Technical University of Denmark, Denmark.ORCID iD: 0000-0001-6492-1245
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2013 (English)In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 95, no 3, p. 357-365Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2013. Vol. 95, no 3, p. 357-365
Keywords [en]
Bacillus anthracis, Genotyping, Luminex, MOL-PCR, SNP, Suspension microarray, genomic DNA, microsphere, oligonucleotide, article, bacterial genome, controlled study, cross reaction, DNA marker, gene technology, genotype, hybridization, limit of detection, molecular probe, multiplex oligonucleotide ligation polymerase chain reaction, nonhuman, nucleic acid analysis, phylogeny, priority journal, reproducibility, single nucleotide polymorphism, Laboratory Proficiency Testing, Molecular Typing, Polymorphism, Single Nucleotide, Reproducibility of Results, Sensitivity and Specificity
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Natural Sciences
Identifiers
URN: urn:nbn:se:ri:diva-39008DOI: 10.1016/j.mimet.2013.10.004Scopus ID: 2-s2.0-84887000414OAI: oai:DiVA.org:ri-39008DiVA, id: diva2:1324973
Note

Tradenames: Luminex xMAP technology, Luminex, United States; Luminex xTAG technology, Luminex, United States; Manufacturers: Luminex, United States; Funding details: European Commission; Funding text 1: The authors thank Joakim Ågren (SVA, Sweden) and Miriam Koene (CVI, Netherlands) for providing DNA samples, as well as Pia Engelsmann (DTU, Denmark) 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 author, and the European Commission cannot be held responsible for any use which may be made of the information contained therein.

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

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Löfström, Charlotta

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