The extraction of DNA is a critical step for species identification by PCR analysis in processed food and feed products. In this study, eight DNA extraction procedures were compared—DNeasy Blood and Tissue Kit, DNeasy mericon Food Kit, chemagic DNA Tissue 10 Kit, Food DNA Isolation Kit, UltraPrep Genomic DNA Food Mini Prep Kit, High Pure PCR Template Preparation Kit, phenol—chloroform extraction, and NucleoSpin Food—Using self-prepared samples from both raw and heat-processed and/or mechanically treated muscles and different types of meat products and pet food (pork, beef, and chicken). The yield, purity, and suitability of DNA for PCR amplification was evaluated. Additionally, comparisons between the effectiveness of various extraction methods were made with regard to price, and labor- and time-intensiveness. It was found that the DNeasy mericon Food Kit was the optimal choice for the extraction of DNA from raw muscle, heat-treated muscle, and homemade meat products from multiple and single species.
This study was conducted to develop systems for the identification of four tuna species (skipjack tuna Katsuwonus pelamis, yellowfin tuna Thunnus albacares, bullet tuna Auxis sp. and Atlantic bonito Sarda sp). At first, raw samples of these species and a mix intended as internal control were prepared for the authentication of fish muscle tissue of the genus Thunnus sp., Auxis sp. and Sarda sp. DNA from raw muscle tissue, the mix and samples was extracted with the DNeasy mericon Food Kit (Qiagen GmbH, Hilden, Germany). The concentration and purity of DNA in raw samples were evaluated using a spectrophotometer. Primers and probe sequences were specifically designed to identify the selected species. In addition, primers and a probe for the endogenous 12S rRNA gene were designed to determine the presence of amplifiable fish (especially tuna) DNA in samples. Furthermore, the species specificity of the designed primers and probes was verified in DNA samples of various tuna and bonito species. Limit of detection for the selected species was calculated as well as the coefficient of determination R2 and efficiency of real-time PCR testing was determined. To evaluate the developed real-time PCR methods, 70 commercial tuna products were analysed. The results show that mislabelling of fish products can still be encountered and, moreover, the presence of an additional species can be identified.
Tuna species are a popular food among consumers. They are mostly sold as heat-processed canned products on the market. Different quality and price of tuna species can lead the producer to the adulteration of food products. The main difficulties in developing a method for species identification in these fish is the high similarity of DNA sequences among close relative fish species. All complete mitochondrial DNA sequences of skipjack tuna (Katsuwonus pelamis) and yellowfin tuna (Thunnus albacares) were compared to all other mitochondrial DNA sequences of tuna fish deposited in the GenBank. The most variable regions within species were determined and primers and probes were designed in this region for the species-specific DNA amplification of skipjack tuna and yellowfin tuna. Moreover, to check the content of amplifiable DNA of fish (namely tuna) in the sample, primers and a probe of mitochondrial 12S rRNA gene in the region of conservative sequence were designed. Real time PCR methods were verified by investigating 51 samples of canned tuna with the declared content of tuna species from the market; the species was confirmed in all tested samples. This method was designed to be suitable for the determination of DNA sequences especially in highly heat treated products.
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