PNNL

Abstract

Multiplexed digital PCR (dPCR) was employed to determine the relative abundance of plasmids pXO1 and pXO2 and chromosomal DNA of 3 strains of Bacillus anthracis: Ames (MLVA cluster A3b), 1035 (South Africa, MLVA cluster B1), and Canadian bison (MLVA cluster A1a). Published multiplexed quantitative PCR assays targeting genes on the chromosome and plasmids pXO1 and pXO2 were evaluated in a digital PCR format using the Fluidigm BioMarkHD™ system. Relative abundances of each species (chromosome and the two virulence plasmids) demonstrated a linear response when tested at five concentrations from 115 expected total DNA molecules to 1,150 expected total DNA molecules for all 3 assays, and for the 3 different Bacillus anthracis strains. With the exception of B. cereus 9241 for the pXO1 assay only, no amplification of near neighbors and no template controls were observed. The ratio of pXO1 plasmid copies to chromosome was approximately 3-4:1, 3:1, and 2:1 for Ames, 1035, and Canadian bison strains, respectively. The ratio of pXO2 plasmid copies to chromosome was approximately 2:1, 2:1, and 1:1 for Ames, 1035, and Canadian bison strains, respectively. The implication for trace detection of virulent B. anthracis in environmental samples is that either plasmid may be detected, but all three confirming targets may not. Because B. cereus and other near neighbors have genetic sequences that are homologous to both pXO1 and pXO2 in B. anthracis, detection of either chromosome, or pXO1, or pXO2, alone, will not be sufficient to confirm virulent B. anthracis in environmental samples.