Single Nucleotide Polymorphisms (SNP)-specific Quantitative Real Time Polymerase Chain Reaction (PCR) Assay for Analyzing Competition and Emergence of the Military Hypersporulating Strains of Bacillus Atrophaeous var. Globigii.

摘要

The utility of single nucleotide polymorphisms (SNPs) for detecting phenotype-specific subpopulations is increasing with the increased understanding of the role of SNPs in adaptation. Based on the recently identification of the Spo0FH101R mutation as phenotype-defining for the hypersporulating military strains of B. atrophaeus, we are developing an assay to detect and quantify hypersporulating military strains of Bacillus atrophaeus var globigii (BG) from mixed cultures with ancestral low spore yield Bacillus atrophaeus variants using the (C:T) SNP corresponding to the Spo0FH101R mutation. The B. atrophaeus congenic pair Detrick-1: Detrick-2 is used for assay validation. The assay is internally calibrated and does not require separate internal-control polymerase chain reaction (PCR), making it high-throughput compatible. Assay specificity 1:1000 is achieved, meaning one spore/cell of Detrick-2 bacteria is detected in the background of 1000 Detrick-1 spores/cells. A novel approach is proposed for analyzing competition experiments based on the relative PCR quantification method and is tested in a hypothetical experiment of emergence of either Detrick-1 or Detrick-2 using mixed bacterial cultures with varying strain frequencies. Strain emergence, characterized by the rate of change in frequency, is calculated from PCR data using our approach. The fitness parameters calculated from the experiments are in good agreement with the theoretical fitness parameter, thus validating the approach's utility.