Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 99 Sensitive GMO detection To determine whether the limit of detection (LOD) of the QIAxcel System is sufficient for GMO testing, template DNA of GMO origin (35S promoter) was mixed with genomic DNA from non-modified plants. Amplification of the GMO band was detected using the QIAxcel System (Figure 1) and quantification was performed to determine the sensitivity of detection (Figure 2 and Table 1). Figure 1. Amplification of target DNA mixed with genomic DNA from non-modified plants. Genomic DNA (supplied by University of Tennesee) was purified from young dogwood leaves using the DNeasy® Plant Mini Kit and quantified with a TD-360 fluorometer (Turner Designs, Sunnyvale, USA). The integrity of the genomic DNA was examined by visual inspection on a 1% agarose gel. Control 35S DNA template (provided by GeneScan, Freiberg, Germany) was mixed with the plant genomic DNA to the final concentrations listed in Table 1. Reactions were prepared with 50 ng/μl of the genomic DNA mixtures and proprietary primers (provided by Biotools, Madrid, Spain). PCR was performed on an Mastercycler® Gradient (Eppendorf, Hamburg, Germany): after an initial denaturation step (94°C for 3 minutes), 40 PCR cycles (denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 40 seconds) were followed by 1 step of 72°C for 3 minutes. PCR products were analyzed in the automatic multi-capillary electrophoresis QIAxcel System using the QIAxcel DNA Screening Kit. Non-diluted PCR products were placed in the instrument sample tray. The DNA samples were automatically injected into the capillary channel and subjected to electrophoresis according to the standard protocol. Separation was performed by the AM320 method using 10 seconds injection time and 320 seconds separation time. A. The expected 225 bp band amplified from the 35S promoter is clearly visible in the gel image. M: The molecular weight and concentration of the amplicons were determined based on the standard pUC18/HaeIII DNA size marker (Sigma, St. Louis, USA). B. BioCalculator software labeled the integrated peak area automatically. The added 35S target DNA was detected at a level of 0.25% in the electropherogram. A B % GMO content 4 2 41 0.5 0.25 0.125 0.06 0 M 225 bp Relative units 2.0 2.5 4.0% 2.0% 1.0% 0.5% 0.25% 0% 3.0 42410.50.250.1250.060 M 2.02.5

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