A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

• 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.

Nyckelord

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

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)