Novel approach for the simultaneous detection of DNA from different fish species based on a nuclear target: quantification potential
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The development of DNA-based methods for the identification and quantification of fish in food and feed samples is frequently focused on a specific fish species and/or on the detection of mitochondrial DNA of fish origin. However, a quantitative method for the most common fish species used by the food and feed industry is needed for official control purposes, and such a method should rely on the use of a single-copy nuclear DNA target owing to its more stable copy number in different tissues. In this article, we report on the development of a real-time PCR method based on the use of a nuclear gene as a target for the simultaneous detection of fish DNA from different species and on the evaluation of its quantification potential. The method was tested in 22 different fish species, including those most commonly used by the food and feed industry, and in negative control samples, which included 15 animal species and nine feed ingredients. The results show that the method reported here complies with the requirements concerning specificity and with the criteria required for real-time PCR methods with high sensitivity.
KeywordsReal-time PCR Nuclear DNA Fish DNA Quantification Rhodopsin gene Degenerate primers/probe
The authors would like to thank Jose Manuel Gallardo and his group from the Marine Research Institute (IIM) of the Spanish National Research Council (CSIC) (Vigo, Spain) for kindly donating well-characterized hake species and Eugénia de Andrade Silva and the Reference Materials Unit of the Institute for Reference Materials and Measurements for their help with the DNA sequencing.
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