Development, Validation and Implementation of a Quadruplex Real-Time PCR Assay for Identification of Potentially Toxigenic Corynebacteria.

Development, Validation and Implementation of a Quadruplex Real-Time PCR Assay for Identification of Potentially Toxigenic Corynebacteria. J Med Microbiol. 2016 Nov 01;: Authors: De Zoysa A, Efstratiou A, Mann G, Harrison TG, Fry NK Abstract Toxigenic corynebacteria are uncommon in the UK, however laboratory confirmation by the national reference laboratory (NRL) can inform public health action according to national guidelines. Standard phenotypic tests for identification and toxin expression of isolates can take from ≥24 h to ≥48 h, from receipt. To decrease the time to result, a real-time PCR (qPCR) assay was developed for confirmation of both identification of Corynebacterium diphtheriae and Corynebacterium ulcerans/ Corynebacterium pseudotuberculosis and detection of the diphtheria toxin gene. Target genes were the RNA polymerase beta subunit-encoding gene (rpoB) and A-subunit of the diphtheria toxin gene (tox). Green fluorescent protein DNA (gfp) was used as an internal process control. Real-time PCR results were obtained within 3-4 hours after receipt of isolate. The assay was validated according to published guidelines and demonstrated high diagnostic sensitivity (100 %), high specificity (98-100 %) and positive and negative predictive values of 91-100 % and 100 % respectively, compared to both block-based PCR and the Elek test, together with a greatly reduced time from isolate receipt to reporting. Limitations of the qPCR...
Source: Journal of Medical Microbiology - Category: Microbiology Authors: Tags: J Med Microbiol Source Type: research