Improved quadruplex real-time PCR assay for the diagnosis of diphtheria
| Autor: | Edgar Badell, Marine Pascal, Sophie Guillot, Marie Tulliez, Norman K. Fry, Sylvain Brisse, Leonardo G. Panunzi, Samuel A. Rose, David Litt |
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| Přispěvatelé: | Biodiversité et Epidémiologie des Bactéries pathogènes - Biodiversity and Epidemiology of Bacterial Pathogens, Institut Pasteur [Paris] (IP), Public Health England [London], This work was supported by Institut Pasteur, Public Health France and Public Health England., We acknowledge the help of Melody Dazas and Annick Carmi-Leroy for the microbiological characterization of the isolates., Institut Pasteur [Paris] |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
DNA
Bacterial 0301 basic medicine Microbiology (medical) [SDV]Life Sciences [q-bio] Corynebacterium pseudotuberculosis 030106 microbiology Corynebacterium diagnostic Real-Time Polymerase Chain Reaction Microbiology law.invention 03 medical and health sciences law MESH: Corynebacterium diphtheriae RNA Ribosomal 16S Corynebacterium ulcerans medicine Humans Diphtheria Toxin diphtheria ComputingMilieux_MISCELLANEOUS Polymerase chain reaction MESH: Diphtheria Diphtheria toxin Corynebacterium diphtheriae MESH: Humans Corynebacterium Infections biology MESH: Real-Time Polymerase Chain Reaction Diphtheria General Medicine medicine.disease biology.organism_classification 16S ribosomal RNA MESH: Diphtheria Toxin Virology MESH: DNA Bacterial 3. Good health MESH: RNA Ribosomal 16S qPCR 030104 developmental biology [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology MESH: Corynebacterium MESH: Corynebacterium Infections real-time PCR |
| Zdroj: | Journal of Medical Microbiology Journal of Medical Microbiology, 2019, 68 (10), pp.1455-1465. ⟨10.1099/jmm.0.001070⟩ Journal of Medical Microbiology, Society for General Microbiology, 2019, 68 (10), pp.1455-1465. ⟨10.1099/jmm.0.001070⟩ |
| ISSN: | 0022-2615 1473-5644 |
| DOI: | 10.1099/jmm.0.001070⟩ |
| Popis: | International audience; 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. |
| Databáze: | OpenAIRE |
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