As part of the Labelfish Project’s work to broaden interest in, and deepen the knowledge of, stakeholders in seafood labelling, traceability and molecular techniques, please find below the second edition of a series of brief reviews of papers in the area. These papers are salient works in terms of their recency or their importance to the area of conservation genetics.
Griffiths, A. M., Sotelo, C. G., Mendes, R., Pérez-Martín, R. I., Schröder, U., Shorten, M., Silva, H. A., Verrez-Bagnis, V. & Mariani, S., 2014. Current methods for seafood authenticity in Europe: is there a need for harmonisation? Food Control 45: 95-100.
Mislabelling of food products has recently received a great deal of public scrutiny, but it remains unclear exactly what methods are being utilised in laboratories testing the authenticity of foods. In order to gain insight into the specific area of the analysis of seafood, a questionnaire focussing on the taxonomic groups typically analysed and the techniques utilised was sent to over one hundred accredited laboratories across the UK, Ireland, Spain, Portugal, France and Germany. Forty-five responded positively, demonstrating significant differences in both the species analysed and methods utilised among the countries included in the survey. Indeed, a diversity of methods was employed across laboratories and efforts to harmonise and/or standardise testing were evident only at national scale. This contrasts with the EU wide scale of regulation on seafood labelling, and may lead to inconsistencies in the results produced in countries.
Amoroso, M. G., De Roma, A., Girardi, G., Guarino, A & Corrado, F., 2014. Italian market fish species identification and commercial frauds revealing by DNA sequencing. Food Control 37: 46-50.
This study used both the cytochrome b gene (Cytb) and the cytochrome oxydase subunit I gene (COI) to determine fish species in 58 Italian commercial seafood products and whole fish. The COI method successfully identified each of the whole fish whereas the Cytb method wasn’t able to characterise two of the samples. COI was subsequently used to identify the processed seafood products. The BOLD Identification System database was used to identify the sequences obtained from the COI gene. The results found that two products labelled “cod” actually contained saithe and the fillets labelled as grouper were in fact Greenland halibut. Both these substitutions suggest a financial motivation as the substituted species were less valuable than the species the producst were labelled as. Phylogenetic analysis was carried out (using the CLUSTAL Omega Multiple Sequence Alignment Program) on these samples to ascertain the fraud.
Morisset, D., Štebih, D., Milavec, M., Gruden, K., & Žel, J., 2013. Quantitative analysis of food and feed samples with droplet digital PCR. PloS one 8(5), e62583. doi:10.1371/journal.phone.0062583
In this article the application of droplet digital PCR (ddPCR) technology for quantification of genetically modified maize is described. In contrast to real-time quantitative PCR (qPCR) methods, ddPCR is an end-point PCR method in which the sample DNA is divided into a several thousands or millions of tiny droplets in water-oil emulsion, each featuring zero, one or more copies of sample DNA target. The PCR reaction takes place in each of the droplets individually and after the reaction is finished, the presence or absence of PCR products is detected in each of the droplets separately
(hence the term “digital” PCR) with the use of flow cytometry. With Poisson statistics, it is possible to count back the absolute number of target DNA molecules contained in the original sample, directly from the ratio of positive to total partitions.
Two devices are needed for this method: one device for the partition of the sample, and another for the read-out of the sample droplets after the PCR reaction (QX100 droplet system from Bio-Rad). The PCR reaction can be conducted in a normal PCR cycler and calibration curves are not needed for this method.
In a duplex assay MON810 transgene and hmg maize reference gene copies were detected. The sensitivity of the method was shown to be 5 target DNA copies and thus comparable to qPCR and to chamber digital PCR (cdPCR) assays. The method had a dynamic range of four orders of magnitude, which is greater than that of cdPCR. When compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and greater tolerance to inhibitors. According to the authors, throughput and cost of this method are advantageous relative to those of qPCR for routine GMO quantification. They conclude that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.
For a more global description of ddPCR technology see also: Pinheiro, L. B., Coleman, V. A., Hindson, C. M., Herrmann, J., Hindson, B. J., Bhat, S., & Emslie, K. R. (2011). Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Analytical chemistry 84(2): 1003-1011.
Everstine, K., Spink, J. & Kennedy, S., 2013. Economically Motivated Adulteration (EMA) of Food: Common Characteristics of EMA incidents. Journal of Food Protection 76: 723-735.
This paper is a review of the EMAs reported in journal articles and media since 1980 in 11 food categories, first of which is fish and seafood. In this category, the authors point different types of EMA: species adulteration or swapping (e. g. farmed fish declared as wild), artificially increasing the weight of the product (by overglazing or overbreading), and mislabeling regarding species. In this last type, the authors recall several mislabeling studies in the United States and Canada, where percentages of mislabelling from 25 to 37 were found. In this context, the authors mention the DNA barcoding as a promising method for species identification, as it is fast, reproducible and standardized. This paper evaluates the current risk of EMA incidents, and determines the lack of adequate regulatory systems or testing methodologies.
The authors highlight the need of innovative methods to prevent and detect EMA in the
Bénard-Capelle, J., Guillonneau, V., Nouvian, C., Fournier, N., Le Loët, K. & Dettai, A., 2014. Fish mislabelling in France : substitution rates and retail types. PeerJ PrePrints | http://dx.doi.org/10.7287/peerj.preprints.327v1 Open Access
This study, undergone by a consortium of citizens, journalist, scientists and non governmental organizations, has evaluated the rate of fish mislabelling in France.
Fish-based samples have been collected in fishmonger shops (74 samples), supermarkets (197 samples) and restaurants (100 samples) and sequenced 390 samples of fish either in fillets or prepared meals. The sampling covers 55 different commercial names. The 5 species most consumed in France represented 67% of the sampling whereas none of the remaining 50 species were tested in more than 18 samples. DNA barcoding was based on the COI sequences using the Barcoding of Life Database (BOLD) and the U.S. Food and Drug Administration (FDA) database.
The overall substitution rate is one of the lowest observed for comparable surveys with large sampling in Europe. They detected no case of species mislabelling among the frozen fillets or in industrially prepared meals. The most of the mislabelling cases were detected directly from the sellers. The rate of mislabelling did not differ between species (3.7 %, ci 2.2-6.4%), except for bluefin tuna (rate of substitution of 83%). Despite a very small sample size (n=6), this species stands in sharp contrast with the low substitution rate observed for the other species. No case of substitution was found in industrially processed food like meals (n=67) or frozen fillets (n=33).