Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 87 Materials and methods Nucleic acid purification and PCR ribotyping C. difficile isolates were subcultured on CCF (cycloserin, cephalosporin, fructose) selective plates and incubated anaerobically for 24–48 hours. After harvesting, genomic DNA was isolated using a Chelex 100-based method (3) and PCR ribotyping was performed as described previously (4). After PCR amplification, samples were concentrated by heating at 75°C for 55 minutes, the volume was adjusted to 10 µl with QX DNA Dilution Buffer, and analysis was performed on the QIAxcel system using the “OM500” method and QX Alignment Marker 15 bp/1 kb. PaLoc analysis The 027 sample, which is positive for ribotyping, was used for detection of the tcdA, tcdB, and cdtA/B genes and the tcdC deletion. Gene sequences were amplified using primers which are described elsewhere (1, 5, 6) and amplicons were analyzed on the QIAxcel system using the “0M500” method and QX Alignment Marker 15 bp/1 kb. DNA izing was performed using the QX DNA Size Marker 50 bp/800 bp. Results and discussion C. difficile PCR ribotyping patterns are based on size variations in the 16S–23S intergenic spacer regions of the bacterial rRNA (rrn) operon. Traditionally, analysis of these variations is performed using agarose gel electrophoresis. While this analysis method is easy to use and relatively cheap, it also requires long run times as well as significant manual effort to pour and prepare gels and often provides poor resolution. Recently, the use of methods based on capillary electrophoresis has been described to discriminate between different C. difficile strains and to analyze infection clusters (7–9). The QIAxcel system is a capillary electrophoresis system that processes sample in batches of 12 and allows analysis of up to 96 samples without manual intervention. The system displays data as both a gel-like image and electropherogram. The QIAxcel system was used for analysis of C. difficile reference strains, and the typical ribotype patterns can be observed in the gel view (Figure 1A and 1B). Comparison of the 027 sample with classical agarose gel electrophoresis reveals a comparable fragment pattern (Figure 1C). The QIAxcel system also proved to be highly suitable for PCR-based detection of the tcdA, tcdB, and cdtA/B genes (Figure 2A). The 18 bp deletion of the tcdC gene was accurately detected by the QIAxcel BioCalculator Software (Figure 2B). A B C Figure 1. C. difficile ribotyping. A. Gel and B. electropherogram views of the 027 ribotype pattern obtained with the QIAxcel system. Alignment markers are indicated in red. C. Comparison of the 027 ribotype pattern obtained with the QIAxcel method (left) and with the traditional agarose gel method (right).

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