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Diphtheria is caused by toxigenic strains ofCorynebacterium diphtheriae, Corynebacterium ulceransandCorynebacterium pseudotuberculosis. For diagnostic purposes, species identification and detection of toxigenic strains (diphtheria toxin (tox)-positive strains) is typically performed using end-point PCR. A faster quadruplex real-time PCR (qPCR) was recently developed (De Zoysaet al. J Med Microbiol. 2016 65(12):1521-1527). Here, we present an improvement of the quadruplex method, in which a 16S rRNA gene target was added as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays. This improved qPCR method was validated using 36 bacterial isolates and 16 clinical samples. The method allows detection of thetoxgene and distinguishingC. diphtheriae(including the newly described speciesC. belfantii) fromC. ulceransandC. pseudotuberculosis. Complete diagnostic specificity, sensitivity and experimental robustness of the method to temperature and reagent concentration variations were demonstrated. The lower limit of detection forC. diphtheriae, C. ulceransandtoxtargets was 1.86 genome copies per 5 μL reaction volume. Finally, the method was successfully used on two distinct qPCR technologies (LightCycler 480, Roche Diagnostics and Rotor-Gene Q, Qiagen) and in two laboratories (Institut Pasteur, Paris, France and Public Health England – National Infection Service, London, UK). This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria. |
