Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 103 Materials and methods The analysis of 8 samples, from sample grinding to identification, was performed in less than 6 h. Samples were ground for homogenization and then lysed via chemical (Buffer ATL), thermal (up to 65°C) or mechanical (1400 rpm agitation) treatment for up to 1 h. DNA was then extracted and purified using the QIAsymphony® DSP DNA Mini Kit on the QIAsymphony. Cytochrome B was amplified with TopTaq Master Mix to yield a fragment of 470 bp. The fragment was digested with a panel of suitable enzymes (AluI, HaeIII, HinfI, Ddel, and TaqI) and the resulting fragments were separated on the QIAxcel Advanced using the QIAxcel DNA High Resolution Kit, the OM500 method, QX Alignment Marker 15 bp/600 bp and QX DNA Size Marker 25–500 bp. Fragment sizes were estimated with the QIAxcel ScreenGel software and results were interpreted from the resulting Excel spreadsheets by comparison to a database of possible profiles. The database contained matching internal and published data. The internal data were validated by analyzing fresh fish with all enzymes at 3 different times, and the published data were validated by testing fish samples with our method. Results and discussion This validation study examined PCR-RFLP for routine identification of fish species, generating results within 8 h. Several fish species, fish mixtures and processed samples were used to validate the method. Our assessment took into consideration that the validation may be limited by either polymorphisms or point mutations causing false negatives and false positives. We also accounted for low-quality food samples, given that overly degraded DNA, even from barely processed food, cannot be analyzed. Table 2 lists the 34 fish species (salmons, tuna, trout, hake) that could be identified with this method based on our database. Figure 1 presents an analysis of DNA from crude fish, both a fish mixture and individual fish species. The HinfI digest (top right) can be used as an example to illustrate the principle of the RFLP approach. The fish mixture (lane 1) revealed 5 bands corresponding to different fish species comprising the mixture. These bands appear also in lanes 2–4 containing DNA from the individual fish species. The bands at 55 and 221 bp were also visible in lane 2 for Scomber scombrus. Analytical method Advantages Inconveniences References PCR-RFLP • Enables admixture analysis • Reproducible, sensitive and specific results • Enables analysis of processed samples • Point mutations can induce false positives or negatives • Identification relies on a profiles database • Without automation, analysis is time-consuming 6, 7 qPCR • Produces quantitative results • Enables analysis of processed samples • Detects trace amounts • Reproducible, sensitive and specific results • Detects only prespecified species • Expensive when examining several fish species (requires 1 analysis per species) 8 PCR sequencing (Sanger Method) • No prior knowledge required (internet database) • Delivers high-quality information • Reproducible and specific results • Does not enable admixture analysis • High analysis costs 9 PCR sequencing (NGS) • Enables admixture analysis • Produces quantitative results • No prior knowledge required (internet database) • Delivers high-quality information • Reproducible and specific results • Time-intensive (1 week for results) • High analysis costs 10, 11 Species-specific PCR • Simple method • Enables analysis of processed samples • Reproducible, sensitive and specific results • Detects only prespecified species • Expensive when examining several fish species (requires 1 analysis per species) 12 Table 1. Most common methods used for identification of fish species

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