Strain-specific single-nucleotide polymorphism assays for the Bacillus anthracis Ames strain

Area of Science:

• Genomics and Evolutionary Biology
• Microbial Forensics
• Molecular Diagnostics

Background:

• Bacillus anthracis (B. anthracis) subtyping is critical for diagnostics and forensic analysis.
• Whole-genome sequencing reveals numerous single-nucleotide polymorphisms (SNPs) in B. anthracis.
• Evolutionary stable SNPs are valuable for pathogen subtyping due to the clonal nature of B. anthracis.

Purpose of the Study:

• To identify specific single-nucleotide polymorphisms (SNPs) that define the lineage of the B. anthracis Ames strain.
• To develop a precise and sensitive method for identifying the B. anthracis Ames strain using identified SNPs.

Main Methods:

• Whole-genome sequencing and evolutionary analysis to identify canonical SNPs in B. anthracis.
• Sequencing and real-time PCR (polymerase chain reaction) to validate identified SNPs across diverse B. anthracis isolates.
• Multiple-locus variable number tandem repeat analysis (MLVA) to assess genetic relatedness of isolates.

Main Results:

• Six highly specific SNPs were identified for the B. anthracis Ames strain (four on the chromosome, one on pX01 plasmid, one on pX02 plasmid).
• These six SNPs successfully differentiated the Ames strain from 88 other unique B. anthracis strains.
• Five of the six SNPs distinguished the Ames strain from its close genetic relatives.

Conclusions:

• The identified SNPs are effective evolutionary and genomics-based signatures for the B. anthracis Ames strain.
• Real-time PCR assays utilizing these SNPs allow for specific and sensitive detection of the Ames strain (<100 fg DNA).
• This approach provides a robust method for discovering strain-specific SNPs in B. anthracis for diagnostic and forensic purposes.