Rapid detection of the Clostridium difficile ribotype 027 tcdC gene frame shift mutation at position 117 by real-time PCR and melt curve analysis

• Published in: European journal of clinical microbiology & infectious diseases 2009-08, Vol.28 (8), p.959-962
• Main Authors: Wolff, D, Brüning, T, Gerritzen, A
• Format: Article
• Language: English
• Subjects: Bacterial diseases; Bacterial diseases of the digestive system and abdomen; Bacterial Proteins - genetics; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Clostridium difficile; Clostridium difficile - genetics; Clostridium difficile - isolation & purification; Deoxyribonucleic acid; DNA; DNA Primers - genetics; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Enterocolitis, Pseudomembranous - diagnosis; Enterocolitis, Pseudomembranous - microbiology; Frameshift Mutation; Human bacterial diseases; Humans; Infectious diseases; Internal Medicine; Medical Microbiology; Medical sciences; Polymerase Chain Reaction - methods; Repressor Proteins - genetics; Ribotyping; Sensitivity and Specificity; Sequence Analysis, DNA; Toxins; Transition Temperature
• ISSN: 0934-9723; 1435-4373
• DOI: 10.1007/s10096-009-0731-7

The emergence of the hypervirulent strain Clostridium difficile PCR ribotype 027 has increased the necessity for rapid C. difficile typing tests for clinical and epidemiological purposes. We developed a rapid real-time polymerase chain reaction (PCR) test for the detection of C. difficile. As the target, we chose the tcdC gene, which encodes for a negative regulator in toxin production. A deletion at position 117 of the tcdC gene, which is associated with severe tcdC truncation, is well conserved in all PCR ribotype 027 isolates. Probe sequences of the real-time PCR test were designed to result in distinct melt profiles for sequence variations at positions 117 to 120 of the tcdC gene. The tcdC gene deletion at position 117 was easily detected with real-time PCR and melt curve analysis in all C. difficile ribotype 027 isolates. In five non-027 strains and 46 hospitalised patient samples, melt curve analysis detected no deletion. PCR results were confirmed by DNA sequencing. The combination of real-time PCR and melt curve analysis is a rapid and accurate method for the detection of C. difficile DNA and simultaneous screening for the tcdC gene deletion at position 117, which is closely related to the C. difficile PCR ribotype 027 strain.