The DNA metabarcoding, based on the Next Generation Sequencing (NGS) technologies, is a promising analytical method to authenticate complex seafood products. However, it is still scarcely applied in the context of the offical control and FBOs self-control systems, mainly due to the lack of standardized workflow protocols (from the sampling to the final data analysis). In this study, the fish burger was selected as experimental model to implement a DNA metabarcoding workflow for the analysis of complex seafood products. A short DNA fragment (≈200 bp) of the 16Sr RNA gene, was selected as molecular target. The sequencing was performed on Illumina platform, and the data were processed using DADA2. The taxonomic assignment was performed using Blastn against GenBank (identity value ≥99%). The inclusion of experimental samples (positive controls, analytical blanks, replicates, samples processed in dirty laboratory environment) throughout the entire workflow was especially taken into account to assess the presence of artefacts/false positive and filter the final data. The PCR protocol, during the library preparation phase, was properly adjusted to limit the presence of primer dimers, probably due to the Illumina overhang adapter sequences. The selected primer pair was proved as good candidate for metabarcoding protocols applied to seafood authentication, although additional performance tests on targeted species should be performed. Sequences of sea bass (Dicentrarchus labrax) were highly predominant in all the analyzed fish burgers, with percentages ≥99.34% except for one, considered as mislabelled, where also many sequences of Atlantic salmon (Salmo salar) were found (12.41%). A threshold value was fixed to remove false-positive sequences (FP) linked to sample crosscontaminations and/or environmental contaminations (e. g. human DNA) at various level of the seafood chain. Overall, the laboratory procedures seemed to minimally contribute to the presence of FP. DNA metabarcoding, beside being confirmed as efficient method to authenticate complex seafood products, was also proved as effective for the evaluation of FBOs management respect to hygiene and safety criteria. However, it is still pivotal to harmonize protocols by adopting measures to be assumed as quality control of the process. This, allowing a more extensive use of metabarcoding in the context of both official controls and FBOs self-control, could increase the capability to reduce food frauds.
Implementation of a DNA metabarcoding workflow for seafood authentication.
Giusti A.
;Malloggi C.;Lonzi V.;Tinacci L.;Armani A.
2023-01-01
Abstract
The DNA metabarcoding, based on the Next Generation Sequencing (NGS) technologies, is a promising analytical method to authenticate complex seafood products. However, it is still scarcely applied in the context of the offical control and FBOs self-control systems, mainly due to the lack of standardized workflow protocols (from the sampling to the final data analysis). In this study, the fish burger was selected as experimental model to implement a DNA metabarcoding workflow for the analysis of complex seafood products. A short DNA fragment (≈200 bp) of the 16Sr RNA gene, was selected as molecular target. The sequencing was performed on Illumina platform, and the data were processed using DADA2. The taxonomic assignment was performed using Blastn against GenBank (identity value ≥99%). The inclusion of experimental samples (positive controls, analytical blanks, replicates, samples processed in dirty laboratory environment) throughout the entire workflow was especially taken into account to assess the presence of artefacts/false positive and filter the final data. The PCR protocol, during the library preparation phase, was properly adjusted to limit the presence of primer dimers, probably due to the Illumina overhang adapter sequences. The selected primer pair was proved as good candidate for metabarcoding protocols applied to seafood authentication, although additional performance tests on targeted species should be performed. Sequences of sea bass (Dicentrarchus labrax) were highly predominant in all the analyzed fish burgers, with percentages ≥99.34% except for one, considered as mislabelled, where also many sequences of Atlantic salmon (Salmo salar) were found (12.41%). A threshold value was fixed to remove false-positive sequences (FP) linked to sample crosscontaminations and/or environmental contaminations (e. g. human DNA) at various level of the seafood chain. Overall, the laboratory procedures seemed to minimally contribute to the presence of FP. DNA metabarcoding, beside being confirmed as efficient method to authenticate complex seafood products, was also proved as effective for the evaluation of FBOs management respect to hygiene and safety criteria. However, it is still pivotal to harmonize protocols by adopting measures to be assumed as quality control of the process. This, allowing a more extensive use of metabarcoding in the context of both official controls and FBOs self-control, could increase the capability to reduce food frauds.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
