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

FORLANI LUCAS; POSIK D. M; BRUNO MC; ZAPPA ME; CASTILLO N S; BARBISAN GISELA; VILLEGAS CASTAGNASSO E E; CRESPI JA; BARONI, VERONICA; WUNDERLING D; PERAL GARCIA P; GIOVAMBATTISTA G

Córdoba

Congreso; VIII Congreso Internacional de Ciencia y Tecnología de los Alimentos; 2022

DNA metabarcoding assay is increasingly used forspecies authentication in industrialized food. This methodological approach,which combines high throughput sequencing strategies with DNA barcoding, allowsto sequence millions to billions of DNA reads from hundreds of samples in asingle run, making it possible to identify all species of interest in complexfoods, including those not declared in the label. The efficiency and outcome ofDNA metabarcoding are dependent upon the design and selection of appropriatemolecular marker sets, as well as on the accuracy of the reference databasesused. This study aimed to evaluate insilico the feasibility of developing a comprehensive method to authenticatethe species of origin in food products, based on targeted next-generationsequencing (NGS). We first selected 177 animal and vegetal species used in thefood industry in Argentina. Some species indicatives of poor manufacturingpractice 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 asmolecular markers. The DNA sequences of these barcoding genes were downloadedfrom the GenBank database (National Center for Biotechnology Information,NCBI). From these sequences, multiplealignments were constructed for each species. A conserved region from eachalignment containing 400 pb was selected. This analysis resulted in a total of336 target regions. The theoretical specificity of this sequence was checkedwith nBLAST against the GenBank database. Then, primer pairs for each region weredesigned using the AgriSeq tools (Thermo FisherScientific, USA). Finally, in silico polymerase chain reactions(PCR) were performed to validate the performance of the designed primers, usingthe nucleotide (nt) database from NCBI and 64 reference genomes. The 336 selected regions showed identities higher than98% with the targeted taxa. However, primers could be designed for only 319 markers.Considering in silico PCRperformance, the markers were classified into four groups: A)  markers that had cross amplification which atleast another reference genome (N = 25); B) markers that amplified the samegenus in the nt database and/or 64 reference genomes (N = 135); C) markers thatamplified another genus in the nt database but did not amplify any genus in thelist of 177 species (N = 128), and D) markers that amplified another genus inthe nt database and some amplified cross genus in the list of selected taxa (N= 31). It is expected that the design ofmultiple primer sets, and a custom database will assist in the selection offood samples for DNA metabarcoding-based analysis, and consequently support thetraceability of commercial foods.