Development of a DNA Metabarcoding Method for the Identification of Crustaceans (

Seafood is a valuable commodity with increasing demand, traded for billions of USD each year. The volatility in supply chains and fluctuating prices contribute to the susceptibility of the seafood market to food fraud. Analytical methods are required to identify seafood in processed foods to ensure food authenticity and compliance with European laws. To address this need, we developed and validated a DNA metabarcoding method for the authentication of crustaceans and cephalopods in processed food samples, as both are prone to food fraud, especially in mixed products. A ~200 bp barcode of the mitochondrial 16S rDNA was selected as the marker for identification and sequenced on Illumina platforms. The DNA metabarcoding method utilizes two primer systems, one for the amplification of crustacean DNA and another for cephalopods. The crustacean primer system comprises two forward and two reverse primers, while the cephalopod primer system includes three forward and one reverse primer. DNA extracts from reference materials, model foods, processed foodstuffs, and DNA extract mixtures were investigated. Even species with a close phylogenetic relationship were successfully identified and differentiated in commercial samples, while single species were detected at amounts as low as 0.003% in model foods. However, false-negative results were obtained for certain species in DNA extract mixtures, which are most likely due to degraded or low-quality DNA and can best be prevented by optimized DNA extraction procedures. Our DNA metabarcoding method demonstrates strong potential as a qualitative screening tool in combination with other in-house DNA metabarcoding methods for food authentication in routine analysis.