Application Guide - QIAxcel Advanced

102 QIAxcel Advanced Application Guide 10/2016 Fish species identification using PCR-RFLP and the QIAxcel® Advanced R. Cassier and C. Compagnon ADGENE Laboratoire, Thury Harcourt, France Introduction Fishing and aquaculture play a considerable economic role in the European Union (1), accounting for 24% of the worldwide fish market (52.2 billion € for 12.3 million tons in 2011). In the EU, aquaculture is regulated through the definition of feed allowed in aquaculture production, including clear restrictions regarding the use of PAP (processed animal proteins), by-products from healthy animals developed to feed other animals (2). Intra-species feeding or cannibalism is prohibited, which means that feeding salmon with Salmonidae species, for example, is forbidden. These regulations are crucial in controlling fish feed composition and ensuring a supply of correctly labelled, high-quality fish to consumers. Despite a long history of regulations to maintain a healthy fish industry, fraudulent practices still exist. One of the most common is the mislabeling of fish species. An initial study in the US between 2010 and 2012 showed that 33% of fish products were mislabeled. The study covered 1200 seafood products originating from 674 retailers and revealed fraud involving mainly red snapper and tuna (3). A similar study in France, based on 371 samples, showed that 3.5% of fish were mislabeled. Fraud involved mainly tuna and cod (4, 5). These studies attest to the importance of controlling all stages of fish distribution. Various techniques are used for fish species identification. The most commonly used is PCR-RFLP (restriction fragment length polymorphism), but other methods, including qPCR, sequencing, and species-specific PCR, are also used (Table 1). PCR-RFLP is a well-documented, easily mastered and inexpensive technique. However, RFLP can be time-consuming and requires special equipment, which makes it a poor candidate for standardized workflows. Furthermore, mutagenic and hazardous products, such as ethidium bromide, are often used in the visualization of DNA sequences. Results from gel electrophoresis can also be complex and interpretation may require specific software, although misidentification can be minimized by maintaining a database of possible profiles. To overcome these problems, we tested the QIAxcel native capillary electrophoresis system as an alternative to conventional gel electrophoresis. QIAxcel Advanced has numerous advantages: analysis is fast (96 samples in 1 h 30 m), inexpensive, and does not require handling ethidium bromide. Additionally, the QIAxcel ScreenGel® software enables semi-automated interpretation. The software calculates size of the analyzed fragments, which can then be interpreted with the ScreenGel software and the Excel® spreadsheets it produces.

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