A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide...

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Single nucleotide polymorphisms (SNPs) are crucial DNA markers for analyzing phylogenetic relationships between bacterial strains.
• High-throughput genotyping methods are needed for efficient SNP analysis in bacterial populations.
• Bacillus anthracis strain typing requires robust and scalable SNP detection methods.

Purpose of the Study:

• To develop an efficient multiplexed SNP genotyping assay for Bacillus anthracis.
• To adapt and optimize the Multiplex Oligonucleotide Ligation-PCR (MOL-PCR) for simultaneous interrogation of 13 phylogenetically informative SNPs.
• To enhance assay performance by reducing cross-reactivity and improving hybridization efficiency.

Main Methods:

• Modification of the MOL-PCR procedure using allele-specific dual-priming-oligonucleotides (DPOs) as MOLigo probes.
• Incorporation of asymmetric PCR for signal amplification and labeling of ligation products.
• Utilized Luminex® xTAG technology for multiplexed SNP detection.

Main Results:

• Developed a 13-plex assay capable of genotyping 13 canonical SNPs in Bacillus anthracis.
• Achieved unambiguous SNP calls and lineage affiliation for 73 representative samples.
• Demonstrated assay limit of detection at 2ng genomic DNA with high reproducibility and robustness across four laboratories.

Conclusions:

• The modified MOL-PCR assay provides a cost-effective, flexible, and scalable solution for Bacillus anthracis SNP genotyping.
• The assay enhances phylogenetic resolution and can be readily adapted for newly identified SNPs.
• Validated proficiency testing confirms the method's reliability for inter-laboratory use.