Application Guide - QIAxcel Advanced

74 QIAxcel Advanced Application Guide 10/2016 Automated MIRU-VNTR genotyping of Mycobacterium tuberculosis strains using the QIAxcel® Advanced Systems M. Gauthier, F. Bidault and J.-L. Berland Emerging Pathogens Laboratory, Fondation Mérieux, Lyon, France Centre International de Recherche en Infectiologie/INSERM U1111, Lyon, France Introduction MIRU-VNTR (mycobacterial interspersed repetitive units– variable number of tandem repeats) genotyping is commonly applied in studies of Mycobacterium tuberculosis strains. The protocol based on mini-satellite allele numbering developed by Supply et al. (1) is widely used. The conventional method for MIRU-VNTR genotyping requires PCR amplification of each targeted locus, followed by detection using high-resolution agarose gel electrophoresis. The allele calling is done manually based on the gel analysis data. Poor gel quality or human error can lead to significant rates of false interpretation. An alternative method is needed to simplify the procedure and increase the reliability of the data assessment to help with the implementation of M. tuberculosis genotyping in high-burden countries. The QIAxcel Advanced System is a high-throughput capillary electrophoresis system with automated data interpretation features. The aim of this study was to assess the instrument’s performance in terms of ease of M. tuberculosis genotyping and reliability of MIRU-VNTR pattern generation. Sizing accuracy, reproducibility, repeatability, and automated allele calling analysis were studied using a MIRU-24 geno- typing panel of the most prevalent strains. Materials and Methods Amplification All of the samples were amplified via PCR using the QIAGEN HotStarTaq® Master Mix Kit, as previously described (2). Two controls were included in each run: one known sample (H37Rv or H37Ra) and one PCR blank (molecular-grade pure water). Conventional method for electrophoresis and allele calling Electrophoresis was run on NuSieve® 3:1 3% Agarose 100 mL Gel for 5 h at 120 V in the presence of the intercalating agent and using several size markers per gel. Allele calling was manually calculated using theoretical tables (2) and reported before submission to a free online genotyping database. QIAxcel-based method for electrophoresis and allele calling PCR products were analyzed using a QIAxcel DNA High Resolution Kit on the QIAxcel Advanced System (protocol OM1700). The QIAxcel Alignment Marker 15 bp/3 kb was run simultaneously with all of the samples to frame or delineate DNA fragments with two clearly visible peaks. The QIAxcel DNA Size Marker 100 bp – 2.5 bp 3:13% Agarose 100

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