1. Introduction

Consumer audience changed food landscape, which responds to the rise in veganism, a greater focus on allergens and demand for craft and artisan fare in home and in restaurants. Quality, safety of food and negative effects of bio-industrial production has become a concern for the today’s consumer [1]. Despite many food quality control programs, the safety perception of consumers has decreased significantly [2]. Food sector has been rapidly internationalized, and it’s location is no longer confined to local or regional supply. The products from the retailers and food industries come from all over the world, transforming the food industry towards an interconnected system with a large variety of complex relationships. Hence these developments, governments, both national and international are responding to this by imposing new legislation and regulations to ensure labelling transparency, ethnic origins, public health and economic transaction [3].Traceability has become an essential requirement to ensure the quality of food products that reach the market. Their implementation in the food industry involves the development of control systems of raw materials, from their entry into the chain of production to their marketing, ensuring the quality and reliability of food for both the producer and the consumer. Species identification based on morphological character are possible and even relatively easy where the manipulation is minimal usually those which are sold whole and without transformation processes, both fresh as chilled or frozen [4]. However, in other cases were external morphological characteristics are eliminated during the processing phase, identification is not possible. Currently most classification methods for species identification are mostly focused on the detection/ amplification of a single DNA species based on PCR technology. On the other hand, if the analysis requires the ability to detect a wide variety of different species, would require testing for each specific kind, making it tedious, costly and not always applicable [5].

DNA barcoding is revolutionary to promote greater traceability and transparency, which is a molecular technique based on the DNA-level identification of differences that univocally characterize individual species. The potential of this technique increased with the merge of next generation sequencing (NGS) platforms speeding up the possibility of simultaneously analyzing multiple ingredients from complex matrixes [6]. NGS technology is based on the massive sequencing of DNA, with significant increase in the ability to obtain information of the sequences of individual molecules within a source of complex or degraded DNA. NGS encompasses both massively parallel and single-molecule sequencing, which provides short and long sequencing reads, respectively. The advantages of this technology include scalability, simplicity and less error prone. This technique is still economically viable and capable of producing results in real time. These are the following main types of new generation sequencing applications: (i) determination of the whole genome sequence of a single cultured isolate, which is commonly referred to as ”whole genome sequencing” (WGS) and (ii) ”target sequencing”, directed sequencing of a determined genome region, among other. The target sequencing demonstrates a huge potential in the areas of food safety and control [7]. For this reason, the FDA is now exploring new regulatory approaches to ensure that NGS tests have adequate analytical performance of the complexity and enormous potential for useful information to generate this new technology. With this approach, we can rapidly identify the genomic information in a given sample [8]. Thus, NGS can be used against problems in food industry, customize internal reporting based on top-of-the-line science and business practices. Hence, this study will demonstrate the efficiency of NGS-based DNA metabarcoding targeted to identify the species presented in food labels not only in with plant, meat, fish, crustacea species [9]. Regarding the fish, meat and crustacea analysis, this study considers high throughput sequencing of different fragments from barcoding genes namely Cytochrome oxidase I (COX I) and Cytochrome b (Cyt b). Whereas the plant approach considers different fragments from different barcoding genes namely Maturase K gene (MATK), Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (RBCL) and Internal Transcribed Spacer 1 (ITS1).

2. Materials and Methods

2.1 Sample preparation and analysis

Samples used in this type of analysis were purchased from local retailers. The authenticity of the samples was verified by Sanger sequencing of mitochondrial gene. The analysis of the sequences of specific organisms has as a target, mitochondrial genes for animal species (COI and Cytochrome B) and for vegetable species chloroplast genes such as (matK, rbcl and trnH-psbA). Apart from that, the present study also identifies a group of species by genes. The method is based on the high throughput sequencing different fragments from barcoding genes.

Analyzed samples were previously homogenized to have better reliability of the results. This step is necessary and applicable by grinding/milling. The amount necessary to be analyzed should be adjusted according to the instructions of the DNA extraction kit used, DNeasy^® mericon^® Food QIAcube^® HT Protocol, Quiagen. The kit DNEasy Mericon 96 QIAcube HT Kit is used for the DNA purification of DNA from a variety of processed matrices, minimizing the transition from PCR inhibitors inherent in complex food samples. The DNeasy mericon 96 QIAcube HT Kit uses modified cetyltrimethylammonium bromide (CTAB) with proteinase K. To the 350μl samples, 350μL of CTAB buffer.

DNA extracted from the test samples are subjected to PCR for amplification of barcoding regions (developed internally). Three microliters DNA template was added to the PCR mix for a total volume of 25 μL. The PCR protocol includes an initial denaturation at 95°C for 2 min, followed by 45 cycles at 95°C for 30s, 48°C for 30 s, and 72°C for 30s. For terminal extension, the reaction was heated for 5 min at 72°C.

DNA library was created with equal amounts of mixed PCR products. The libraries were purified with AgentCourt^® AMPure^® XP beads considering manufactures instructions. Qubit™ dsDNA HS Assay Kit was used to quantify the final library. Purified amplicons were then used for the preparation of the amplicon libraries for subsequent NGS sequencing in the Ion torrent system (illustrated in Figure 1). Generically, purified amplicons are subjected to End Repair reactions followed by ligation of the sequencing adapters, and nick repair to complete the bond between adapters and inserts. Finally, the amplification of the libraries, the procedure is schematically illustrated below.

Figure 1: NGS analysis worfow.

The sequences obtained from Chef™ System and Ion PGM™ Systems (Thermo Fisher Scientific Inc.) [4]. A specific bioinformatic analysis pipeline was specifically and internally constructed to process the large amount of sequences generated by the NGS technology which includes grouping identical sequences and matching with genetic data bases for species identification.

3. Results

3.2 Sample analysis

This simple approach matches and identifies more than 20 different types of food labelling. The samples, which some of them were pure and others contained mixtures of different tissues, lead to the correct identification of the intended species. The BLASTn analysis produces results were the most significant results were the ones with the lowest E-value. The lowest E-value represents the most accurate matching during the blast analysis [16]. The BLASTn results were parsed to count how many times a specie was identified, according to the input parameters. The results of the clustered analysis were included has well. All species detected using DNA sequences obtained using the Next generation DNA sequencing (NGS) methodology could be identified to the species level to the threshold of 98% of similarity against a curated reference DNA library. We have analyzed 342 fish samples, 482 meat samples, 35 crustacea samples, 59 plant samples. Table 1 lists some of the species analyzed by target sequencing, with the %ID of each species.

Table 1: Summary of the "pure" samples analyzed, with the respective analysis pipeline identified. Since the plant analysis, has a lot of pure samples, this table presents some species.

The limit of detection was determined using mixtures of gDNA from different species in different DNA concentrations containing 10%, 1%, 0.5% 0.1% of the target species to identify. The obtained results are presented on Table 2. In all cases the obtained limit of detection was 0.5% of target specie in a mixture of other species (either from the same or different group of species).

Table 2: Summary of the mixture’s species analyzed, considering the different pipeline analyzes.

Additionally, food samples, of complex composition were retrieve from the supermarket and analyzed using the developed methodologies. In all the analyzed samples, we were able to identify all the ingredients present on the label (Table 3) confirming that the methods developed are suitable to be put in use for food fraud detection.

Table 3: Summary with the results from the real samples analyzed.

4. Discussion

Species identification resorting to Sanger sequencing has been the gold standard method so far and has been proved a powerful tool. With the development of the new technologies namely the NGS technology based on massive DNA sequencing, with a significant increase in the ability to obtain information on the sequences of individual molecules within a complex or degraded DNA source. The advantages of this technology include scalability, simplicity, being less prone to errors, and even more economically viable with the eventual objective of obtaining results in real time. The number of applications NGS is unlimited. Although there are still many problems to solve, a priori the DNA of any organism can be sequenced. NGS technology can and is currently being applied directly to various sectors such as the clinical diagnosis, forensic analysis and demonstrates also having enormous potential in the areas of security and control to feed. For this reason, the FDA is now exploring new regulatory approaches to ensure that NGS tests have analytical performance suited to the complexity and enormous potential of generating information useful of this new technology.

Animal species identification has been based on a standardized genetic target with DNA barcoding. To the amplification of a target gene, “universal primers” were designed in conserved regions that surround hyper variable regions, with less than 400 bp [17]. With this study, we demonstrate the utility of target-based approach allergen food authentication for meat, fish, plants and vegetable products. Ion S5 system sequencing, is one of the high-throughput sequencing methods, which originated novel ways to detect allergen and control food authenticity.

From the bioinformatic analysis, the number of reads obtained from each species were not the same between the different approaches. This difference is due to PCR bias, sequencing artifacts and other technical challenges. Nevertheless, the developed methodology allows the identification of fish species, crustacea and plant origin allowing, in case they are presented to identify it and the specie, with a detected liming of 0.5%. All the development methodologies allow the species identification presented in samples at a specie level regarding fish and crustacea analysis. As for the plant analysis, the identification was only possible in some cases at the gender level, with a limit of detection of 0.5% (5000 ppm).Specially the mixed samples where several organisms from the same genus are present the analysis is more complex returning several species form the same genus, and in such cases a direct comparison with the product label must be performed in order to ensure the compliance.

Hence, the use of different fragments and improvement of lab techniques have strong chances of reducing the imprecision and resolution of NGS results. Some of them, present unexpected species, in some of the analyzed samples, this is due to sequencing errors or alignment errors. Although the quantification of DNA can be high sometimes, that amount can’t be related to the amount of reads per samples, specially the mixed samples. Thus, the target DNA sequencing method continues to be a powerful tool for authentication and mislabeling detection in several products.

Adulteration in some products include many issues, such as the replacement of the primarily animal species by low-value animal or vegetable species, mislabeling of ingredients and the geographical origin, misunderstood processing methods and the geographical origin, undeclared processing methods, the addition of undeclared ingredients and non-meat components [10]. For samples with processed meat and fish products, the inability to visually identify species in these products, coupled with price variations in different animal species, increases the probability of species substitution [18].

Overall, there are two types of food adulteration, the replacement of a species for another one due to financial issues, and unintentional adulteration due to accidental alteration.

5. Conclusion

The study presents a methodological tool for species identification in different types of samples. The results presented by the abundance of each species, can determine the amount of those in a sample and if, in fact, if they are presented or not. The target sequencing allows the rapid detection of mislabeling, fraud or accidental contaminations. Hence, this project delivers a single analysis using NGS technology, to detect the presence of a certain group or subgroup of the suggested analysis in this study, in foods and / or materials that are in contact with food. This technology allows the identification of all species present even in complex food matrixes with a level of detection of 0.5%.

This technology allows to accurately identify the species (vegetables / plants, fish, crustaceans) present, without the need to carry out various analyzes on a mixture of many ingredients, as well as allowing the detection of unanticipated food components, which was previously impossible. Besides that, this approach is viable in terms of cost-effective when used to analyze several samples and it can also detect DNA in low amounts.

Competing Interests

The authors declare that they have no competing interests.