Identification and molecular discrimination of toxigenic and nontoxigenic diphtheria Corynebacterium strains by combined real-time polymerase chain reaction assays

With the recognition of several diphtheria outbreaks and the emergence of nontoxigenic corynebacteria strains, there has been renewed interest in the development of laboratory diagnostic methods. Previously reported polymerase chain reaction (PCR) assays can have low diagnostic sensitivity or give species misidentifications among clinical isolates. The aim of the present study was the development of combined real-time PCR assays, based on the tox and rpoB genes, for the detection and differentiation of toxigenic and nontoxigenic corynebacteria. By the PCR tox assay, it was possible to perform the direct identification of DT tox gene of Corynebacterium diphtheriae and Corynebacterium ulcerans, while the PCR rpoB assay differentiated C. diphtheriae from C. ulcerans, irrespective of their toxigenic status. In addition, we detected the DT toxin of Corynebacterium pseudotuberculosis for the first time. These assays revealed high sensitivity, specificity, and reproducibility, and the availability of plasmid controls will facilitate further research into the diagnostics of diphtheria corynebacteria.