Design of a next-generation sequencing method for the authentication of industrialized food using DNA metabarcoding analysis

FORLANI L; POSIK DM; BRUNO MC; ZAPPA ME; CASTILLO NS; BARBISAN G; VILLEGAS CASTAGNASSO EE; CRESPI JA; BARONI V; WUNDERLIN D; PERAL-GACÍA P; GIOVAMBATTISTA G

Congreso; VIII Congreso Internacional de Ciencia y Tecnología de Alimentos (CICyTAC); 2022

DNA metabarcoding assay is increasingly used for species authentication in industrialized food.This methodological approach, which combines high throughput sequencing strategies with DNA barcoding, allows to sequence millions to billions of DNA reads from hundreds of samples in a single run, making it possible to identify all species of interest in complex foods, including those not declared in the label. The efficiency and outcome of DNA metabarcoding are dependent upon the design and selection of appropriate molecular marker sets, as well as on the accuracy of the reference databases used. This study aimed to evaluate in silico thefeasibility of developing a comprehensive method to authenticate the species of origin in food products, based on targeted next-generation sequencing (NGS). We first selected 177 animal and vegetal species used in the food industry in Argentina. Some species indicatives of poor manufacturing practice were also incorporated. Cytochrome c oxidase subunit I (COI), mitochondrial cytochrome b (Cytb) and chloroplast RuBisCO large subunit (RbcL) and Megakaryocyte-Associated Tyrosine Kinase (matK) genes were selected as molecular markers. The DNA sequences of these barcoding genes were downloaded from the GenBank database (National Center for Biotechnology Information, NCBI). From these sequences, multiple alignments were constructed for each species. A conserved region from each alignment containing 400 pb was selected. This analysis resulted in a total of 336 target regions. The theoretical specificity of this sequence was checked with nBLAST against the GenBank database. Then, primer pairs for each region were designed using the AgriSeq tools (Thermo Fisher Scientific, USA). Finally, in silico polymerase chain reactions (PCR) were performed to validate the performance of the designed primers, using the nucleotide (nt) database from NCBI and 64 reference genomes. The 336 selected regions showed identities higher than 98% with the targeted taxa. However, primers could be designed for only 319 markers.Considering in silico PCR performance, the markers were classified into four groups: A) markers that had cross amplification which at least another reference genome (N = 25); B) markers that amplified the same genus in the nt database and/or 64 reference genomes (N = 135); C) markers that amplified another genus in the nt database but did not amplify any genus in the list of 177 species (N = 128), and D) markers that amplified another genus in the nt database and some amplified cross genus in the list of selected taxa (N = 31). It is expected that the design of multiple primer sets, and a custom database will assist in the selection of food samples for DNA metabarcoding-based analysis, and consequently support the traceability of commercial foods.