Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 73 For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor. Visit www.qiagen.com/qiaxcel-genotyping and find out how automated gel electrophoresis can benefit your lab! Trademarks: QIAGEN® , Sample to Insight® , QIAxcel® (QIAGEN Group); GeneAmp® (Applera Corporation or its subsidiaries). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. Ordering www.qiagen.com/shop Technical Support support.qiagen.com Website www.qiagen.com Three of the 20 genes assayed were found to be a source of problems in the manual agarose gel-based assay, with one gene prone to false negatives and two genes prone to misclassification. Despite the high concordance between the platforms, fully a third of the samples analyzed (32/96 samples) were affected by problems with these genes and had at least 1 erroneous band call on agarose gel electrophoresis. This would affect our ability to detect strains with 100% matching fingerprints, a key aspect of molecular epidemiological investigations. The data obtained from the QIAxcel was instrumental in identifying these genes so that PCR primers could be re-designed to mitigate these adverse effects. Conclusions Although the QIAxcel displayed extremely high concordance with conventional agarose gel electrophoresis, greater sizing accuracy and greater sensitivity of detection allowed the QIAxcel data to outperform the agarose gel data. The QIAxcel system enables easy maintainance of high quality control over multiple electrophoresis runs and displays a wider dynamic range than conventional gel electrophoresis. As a result, bands both in weak samples and in highly concentrated samples are accurately detected, making the QIAxcel an extremely robust, high-throughput platform for these types of genotyping applications. False negative (i.e “weak”) False positive (i.e “wrong size”) Misclassified False negative (i.e “weak”) False positive (i.e “wrong size”) Misclassified Figure 5. Classification of mismatches observed in the dataset after analysis by agarose gel electrophoresis or on the QIAxcel. Each of the 50 mismatches was examined to determine the nature of the mismatch. False negatives (38%, 19/50) were weak amplicons that were below the detection limit for agarose gel electrophoresis but could be detected on the QIAxcel (see Figure 1). False positives (6%, 3/50) were amplicons of incorrect size and called positives after agarose gel electrophoresis due to sufficient similarity to the expected amplicon size (see Figure 2). Misclassified (56%, 28/50) were amplicons misclassified after agarose gel electrophoresis because they were of sufficiently similar size to the expected bands, e.g., 282 bp compared with 307 bp (see Figure 3). References 1. Taboada, E.N. et. al. (2004) Large-scale comparative genomics meta-analysis of Campylobacter jejuni isolates reveals low genome plasticity. J. Clin. Microbiol. 42, 4566. 2. Zhang, Y. et al. (2007) Genome evolution in major Escherichia coli O157:H7 lineages. BMC Genomics 8, 121. 3. Taboada, E. N. et al., (2008) Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of Campylobacter jejuni. BMC Evol. Biol. 8, 229. 4. Laing, C. et al. (2008) Rapid Determination of Escherichia coli O157:H7 Lineage-Types and Molecular Subtypes Using Comparative Genomic Fingerprinting (CGF). Appl. Environ. Microbiol. 74, 6606.

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