title: Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis.
abstract: Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.
authors.affiliation: Department of Microbiology, College of Medicine, Chung-Ang Univerisity, 221, Heuksuk-dong, Dongjak-gu, Seoul 156-756, South Korea. kimkj@cau.ac.kr
authors.forename: Kijeong
authors.initials: K
authors.lastname: Kim
authors.forename: Juwon
authors.initials: J
authors.lastname: Seo
authors.forename: Katherine
authors.initials: K
authors.lastname: Wheeler
authors.forename: Chulmin
authors.initials: C
authors.lastname: Park
authors.forename: Daewhan
authors.initials: D
authors.lastname: Kim
authors.forename: Seungjoon
authors.initials: S
authors.lastname: Park
authors.forename: Wonyong
authors.initials: W
authors.lastname: Kim
authors.forename: Sang-In
authors.initials: SI
authors.lastname: Chung
authors.forename: Terrance
authors.initials: T
authors.lastname: Leighton
mesh_terms: D001408:Bacillus anthracis / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D001409:Bacillus cereus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D005798:Genes, Bacterial; D010802:Phylogeny; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
chemical_list: D004269:DNA, Bacterial