Improved quadruplex real-time PCR assay for the diagnosis of diphtheria - Institut Pasteur Accéder directement au contenu
Article Dans Une Revue Journal of Medical Microbiology Année : 2019

Improved quadruplex real-time PCR assay for the diagnosis of diphtheria

Résumé

Introduction. Diphtheria is caused by toxigenic strains of Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium 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 Zoysa et al. J.Med.Microbiol. 2016;65(12):1521-1527).Aims. We aimed to improve the quadruplex method by adding a 16S rRNA gene target as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays, thus avoiding the possibility of false-negative reporting.Methodology. Universal 16S rRNA gene primers and a probe were defined. The novel method was tested using 36 bacterial isolates and 17 clinical samples. Experimental robustness to temperature and reagent concentration variations was assessed.Results. The method allows detection of the tox gene and distinguishing C. diphtheriae (including the newly described species Corynebacterium belfantii) from C. ulcerans and C. pseudotuberculosis. Complete diagnostic specificity, sensitivity and experimental robustness were demonstrated. The lower limit of detection for C. diphtheriae, C. ulcerans and tox targets was 1.86 genome copies per 5 µl reaction volume. 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).Conclusion. This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria.
Fichier principal
Vignette du fichier
1455_jmm001070.pdf (597.58 Ko) Télécharger le fichier

Dates et versions

pasteur-03259553 , version 1 (14-06-2021)

Licence

Paternité

Identifiants

Citer

Edgar Badell, Sophie Guillot, Marie Tulliez, Marine Pascal, Leonardo Gabriel Panunzi, et al.. Improved quadruplex real-time PCR assay for the diagnosis of diphtheria. Journal of Medical Microbiology, 2019, 68 (10), pp.1455-1465. ⟨10.1099/jmm.0.001070⟩. ⟨pasteur-03259553⟩

Collections

PASTEUR
120 Consultations
123 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More