Application Guide - QIAxcel Advanced

86 QIAxcel Advanced Application Guide 10/2016 QIAxcel® System — Clostridium difficile ribotype determination Veit Braun,1 Carola Schade,2 Daniel Lehmann,2 and BeeNa Lee2 1 tgcBIOMICS GmbH, Mainz, Germany 2 R&D Department, QIAGEN GmbH, Hilden, Germany Abstract In this application note, we describe the transfer of methods based on agarose gel electrophoresis for ribotype detection and genotype characterization of Clostridium difficile to the QIAxcel system. Using the QIAxcel system, we were able to detect the fragment pattern characteristic of the 027 ribotype C. difficile strain in both gel and electropherogram views. The system also proved highly suitable for PCR-based detection of the tcdA enterotoxin and tcdB cytotoxin genes as well as the tcdC deletion. These loci together with the tcdR and tcdE genes form a chromosomal pathogenicity locus (PaLoc) (1, 2). The cdtA/B binary toxin gene was also detected. Introduction Clostridium difficile is the major cause of nosocomial diarrhoea and antimicrobial-associated colitis. Millions of infections a year cause diarrhoea, sometimes with abdominal pain and vomiting. Enteritis caused by C. difficile infection has become an increasing problem in the past few years. Since 2002, severe outbreaks have been reported with increased mortality for both elderly and young patients. Antibiotics, especially those with a broad-spectrum activity, cause disruption of normal intestinal flora and can lead to overgrowth of C. difficile, which flourishes under these conditions. It is suspected that use of these antibiotics drives the formation of hypervirulent strains. Identified hypervirulent strains are of the 027 ribotype and express 3 toxins: enterotoxin TcdA, cytotoxin TcdB, and binary toxin CdtA/B. Furthermore, a deletion within the regulatory tcdC gene, normally restricting production of the TcdA and TcdB toxins, is characteristic for these strains. We evaluated use of the QIAxcel, a capillary electrophoresis system, for ribotyping C. difficile strains and analysis of toxin genes. In comparison to conventional agarose gel based methods, this system provides significantly shorter run times and fewer manual handling steps, freeing up time for more demanding lab work and reducing manual error rates.

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