title: Development of a real-time fluorescence PCR assay for rapid detection of the diphtheria toxin gene.
abstract: We developed and evaluated a real-time fluorescence PCR assay for detecting the A and B subunits of diphtheria toxin (tox) gene. When 23 toxigenic Corynebacterium diphtheriae strains, 9 nontoxigenic C. diphtheriae strains, and 44 strains representing the diversity of pathogens and normal respiratory flora were tested, this real-time PCR assay exhibited 100% sensitivity and specificity. It allowed for the detection of both subunits of the tox gene at 750 times greater sensitivity (2 CFU) than the standard PCR (1,500 CFU). When used directly on specimens collected from patients with clinical diphtheria, one or both subunits of the tox gene were detected in 34 of 36 specimens by using the real-time PCR assay; only 9 specimens were found to be positive by standard PCR. Reamplification by standard PCR and DNA sequencing of the amplification product confirmed all real-time PCR tox-positive reactions. This real-time PCR format is a more sensitive and rapid alternative to standard PCR for detection of the tox gene in clinical material.
authors.affiliation: Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Mothershed
authors.forename: Pamela K
authors.initials: PK
authors.lastname: Cassiday
authors.forename: Kevin
authors.initials: K
authors.lastname: Pierson
authors.forename: Leonard W
authors.initials: LW
authors.lastname: Mayer
authors.forename: Tanja
authors.initials: T
authors.lastname: Popovic
mesh_terms: D003353:Corynebacterium diphtheriae; D017931:DNA Primers; D004165:Diphtheria / Q000175:diagnosis* / Q000382:microbiology; D004167:Diphtheria Toxin / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004167:Diphtheria Toxin