Digital archive of theses discussed at the University of Pisa


Thesis etd-07052017-140228

Thesis type
Tesi di dottorato di ricerca
Thesis title
DNA-based techniques for the prevention of commercial and health frauds in seafood products: between tradition and innovation
Academic discipline
Course of study
tutor Prof. Armani, Andrea
  • species identification
  • seafood
  • Sanger sequencing
  • NGS technologies
  • frauds
  • DNA-based methods
Graduation session start date
The globalization of the seafood supply chain has progressively led to the presence of an extremely wide gamma of new commercial species (fish and shellfish but also unconventional seafood) as well as to the massive entrance of numerous types of processed products on international markets. The growth in European imports from third countries and particularly from China, which despite its central role in the world’s seafood market effectively lacks a suitable regulation for fishery products, represents an important challenge for international traceability control systems. The impossibility to appeal to visual identification in processed commodities has in fact made easier the fraudulent substitution of high-value products with less expensive or lower quality alternatives, damaging world economy as well as environmental balance. Moreover, the unaware consumer is at risk when health-threatening species are used. Thus, species identification acquires fundamental importance in the prevention of phenomena of counterfeiting and adulteration for consumer protection. DNA-based analytical methods are the most applied for species identification due to the resistance of nucleic acid to physical-chemical treatments and degradation, representing a valuable tool for traceability in different types of products (fresh, processed, canned). However, the most appropriate DNA-based technique to be used should be selected according to the species and the kind of product to be analysed. Present DNA-based techniques can rely on sole PCR amplification, producing directly readable results, or methods requiring a post PCR DNA sequencing step, further divided in the classical and mostly used “first generation sequencing technologies” and in the “Next Generation Sequencing (NGS) Technologies”, less utilized yet but particularly attractive in the food inspection research field. In this perspective, six research projects, that have been grouped in three technical sections, were reported in this doctoral thesis. In the first section two projects not requiring to the sequencing phase (NO-SEQUENCING TECHNIQUES) are included. In the first project a Pentaplex PCR for the identification of five edible jellyfish species (Nemopilema nomurai, Rhopilema esculentum, Rhizostoma pulmo, Pelagia noctiluca, and Cotylorhiza tuberculata) was developed. A common degenerated forward primer and five specie-specific reverse primers were designed to amplify Cytochrome Oxidase subunit I (COI) gene regions of different lengths. Considering the high level of degradation in the DNA extracted from acidified and salted products, the maximum length of the amplicons was set at 200 bp. The assay gave successful amplifications and identification in 85.4% of 48 ready-to-eat products and in 60% of 30 classical salted products collected on the market. In the second project a conventional multiplex PCR for discriminating the toxic species Ruvettus pretiosus and Lepidocybium flavobrunneum (Gemfish), containing high amounts of indigestible wax esters that often provoke gastrointestinal disorders, from other potentially replaced species (tunas, cod, and sablefish) was proposed. A common degenerate forward primer and three species-specific reverse primers were designed to amplify COI gene regions of different lengths of gemfish, tunas, and sablefish, respectively, while a primer pair was designed to amplify a fragment of the cytochrome b (cytb) gene of cod species. The method produced the expected amplicon in all the DNA samples tested (reference and commercial).
The second section includes two projects requiring first generation classical Sanger sequencing (1st GENERATION SEQUENCING TECHNIQUES): one project evaluated the effects on amplification and sequencing performance of two types of oligonucleotide tails (by Steffens and by Messing) added to PCR primers. The evaluation was carried out in silico and also practically assessed on DNA samples of edible species (birds, fishes, and mammals). While 16SrRNA tailed and non-tailed primers performed similarly, differences were found for COI primers. Messing’s tails negatively affected the reaction outputs, while Steffens’ tails significantly improved the band intensity and the length of the final contigs based on the individual bidirectional read sequence. In another project DNA and mini-DNA Barcoding was applied for the identification of commercial products sold with the Chinese term 鳕 (cod) in supermarkets and in the online market in China. The mislabelling rate was assessed according to three increasingly stringent definitions (1. cod meaning Gadiformes species; 2. cod meaning Gadus spp.; 3. Cod not meaning any specific species, since the qualifier “Atlantic”, “Pacific” or “Greenland” must be added in order to refer to Gadus morhua, Gadus macrocephalus or Gadus ogac, respectively) due to the lack of harmonization around the denomination “cod” in China. A very high mislabelling rate, which exceeded 60% even with the less stringent definition, was highlighted.
Finally, two project approaching NGS technologies (NGS TECHNIQUES) were conducted: in one of them seafood species commonly utilized in surimi products preparation were chosen as target for the analysis of 14 pairs of universal primers for the 16SrRNA, cytb and COI mitochondrial genes, in order to assess their potential utilization in NGS analysis. Firstly, an in silico evaluation was conducted in order to test primers coverage capacity on the basis of the number and the position of mismatches within the target sequence available on databases. Then, the 9 primer pairs which showed the best coverage capacity were tested in PCR reaction on the DNA of collected fish and cephalopod species. The primers pair proposed by Chapela et al. (2002) showed the best results in both in silico and PCR analysis. Overall, results confirm that 16SrRNA primers are the most suitable for the NGS analysis finalized to seafood species detection. In the last project NGS techniques were applied to the analysis of surimi sample sproduced in European and Asian countries. Ion Torrent PGM Platform was used to sequence a short 16SrRNA fragment with universal primers (designed by Chapela et al., 2002) able in amplifying DNA from both fish and cephalopod species reported as commonly used in surimi production. After the taxonomic analysis of the obtained reads, a certain degree of species variability was observed, especially in Asian products. 37.5% of the samples were found as mislabelled. Among them, 25% voluntary declared a species that actually not corresponded to that found and 25% not reported at all the presence of molluscs on the label, representing a potential health threat for allergic consumers. Utilization of vulnerable species was also proved. Ion Torrent PGM was proved as an useful tool for the analysis of processed multispecies seafood products, even though the method should be further deepened, particularly regarding the aspect of DNA quantification.