Application Guide - QIAxcel Advanced

70 QIAxcel Advanced Application Guide 10/2016 Advantages of the QIAxcel® system for bacterial genotyping Steven Mutschall,1 Susan Ross,2 Cody Buchanan,1 and Eduardo N. Taboada1 . 1 Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Lethbridge, Alberta, Canada 2 Animal Diseases Research Institute, Canadian Food Inspection Agency, Lethbridge, Alberta, Canada The QIAxcel system was successfully used together with the QIAxcel DNA Screening Kit for high-throughput genotyping of bacteria. The QIAxcel system enabled greater sizing accuracy and more sensitive detection than conventional agarose gel electrophoresis. Introduction Comparative genomic studies have demonstrated extensive intraspecies genomic variability in some bacterial species and have led to identification of “accessory” genes that are present in some but not all strains of C. jejuni and verotoxigenic E. coli (1–3). Comparative genomic fingerprinting (CGF) is a novel method of comparative genomics-based bacterial characterization based on the concept that differential carriage of these accessory genes can be used to generate unique genomic fingerprints for genotyping purposes. A CGF assay for the analysis of E. coli was recently developed in our laboratory and shows great promise as a high-throughput comparative genomics-based method for genotyping that yields epidemiologically relevant information (4). We recently developed a CGF method for C. jejuni based on assessing the conservation status of 20 accessory genes. These 20 genes are targeted by a series of four 5-plex PCRs designed based on data from multiple sequenced genomes. Target genes were selected to represent whole-genome genetic diversity by targeting hyper-variable regions previously identified (1). The genes selected were either present or absent on different genome strains and displayed little sequence variation when present. The latter enabled PCR primers to be easily designed in SNP-free regions. Although the CGF method has a favorable throughput when compared to standard methods for C. jejuni genotyping, we sought to adapt the assay to the QIAxcel system to increase our throughput and facilitate data analysis. As part of this process, we performed extensive cross-validation to compare conventional agarose gel results to those obtained using the QIAxcel instrument. Materials and Methods CGF assay PCR Each gene in the assay is represented by a signature amplicon, with each 5-plex PCR producing a unique 5-band finger- print, and this presence/absence profile of the 20 genes is used to produce the comparative genomic fingerprint. PCR was carried out in a 50 μl reaction volume, with The QIAxcel system enables fully auto- mated analysis of up to 96 samples per run.

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