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.
The most common techniques used to identify tuna species include methods based on the detection of specific DNA. The quality of species-specific DNA crucially affects the efficiency of amplification during the subsequent polymerase chain reaction (PCR). Detection of DNA in processed products can be adversely affected by DNA fragmentation during the processing steps and the use of ingredients that may inhibit the PCR reaction. In this study, several processing treatments applied to the muscle tissue of yellowfin tuna (Thunnus albacares) were evaluated. For DNA isolation three DNA extraction methods were compared. The concentration, purity, and amplificability of DNA were tested. The results revealed the variability among extraction procedures in terms of DNA quality and quantity in tuna muscle tissue processed under different processing technologies. ARTICLE HISTORY
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.
Multiplex PCR analysis for the detection of two targeting segments of genes coding major food protein allergens as peanut (Arachis hypogaea) Ara h 1 gene and hazelnut (Corylus avellana) Cor a 1 gene was developed. Two sets of primers were designed and tested to their specificity on a broad range of ingredients. The identity of amplicons (Ara h 1-180 bp, Cor a 1 -258 bp) by sequencing and alignment of sequences with sequences deposited in Genbank was confirmed. When testing the specificity of designed primer pairs on a spectrum of food ingredients, no cross reactions were detected. A potential inhibition of PCR reaction was eliminated using the universal plant primers of chloroplast gene 124 bp for the plant matrices confirmation. The intrinsic detection limit was 10 pg·ml -1 and the practical detection limit was 0.001% w/w (10 mg·kg -1 ) for both peanuts and hazelnuts. The method was applied to the investigation of 60 commercial food samples. The developed multiplex PCR method is cheap, specific and sensitive enough and can be used as a simple, one day procedure for the checking of undeclared peanut and hazelnut major allergens in food. Plant allergens, DNA, Ara h 1, Cor a 1, food
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