Guest Article: Fish speciation analysis

Following this, combined with the recommendations made by the Chris Elliott report, a demand for authenticity testing has increased.  

A particular area of interest is fish speciation analysis. Increasing consumption has led to the over-exploitation of certain fish species resulting in opportunities for species substitution and food adulteration. 

A study by Oceana in the USA analysed more than 1,000 samples with mislabelling identified in grocery stores, restaurants and sushi venues ¹​.  The greatest number of mislabelling occurred in sushi venues with 74% mislabelled fish samples ¹​. 

In Europe, a recent study highlighted fish mislabelling prevalent in 30% of samples from restaurants in Brussels. This work demonstrated the replacement of fish with cheaper alternatives, examples include Cod and Blue Fin Tuna being replaced with Pangasius and Yellowfin Tuna, respectively ²​.

Analytical help​

Commercial and government laboratories are offering fish speciation analysis to enable food producers, restaurants and retailers to confirm the fish species and identify food fraud. 

Several methods are available including real-time PCR (Polymerase Chain Reaction). Real-time PCR is a sensitive, accurate and rapid method of analysis which targets DNA. This method is able to replicate the target DNA to visualise the presence of the DNA. This can only be used as a targeted approach, therefore only confirming the presence or absence of a requested species.   

A second approach is RFLP (Restriction Fragment Length Polymorphism) and involves 2-stages; firstly, replication of the target DNA using PCR. Secondly, the replicated DNA is then subjected to enzymes which cut the target DNA in certain areas and is dependent on the fish species.

For example, the DNA may be cut into three segments if the DNA belongs to Pacific Cod and into two segments if it belongs to Atlantic Salmon. These are distinguished by analysing the size of the segments of DNA. This allows the analyst to compare the profile with a database to determine the species of the unknown sample.

NGS growth​

Further methodologies are becoming available including DNA sequencing, particularly Next Generation Sequencing (NGS). NGS is a non-targeted approach and is able to identify a mixture of fish species present in a sample.

This method exploits the fish DNA sequence which differs between species and is made up of a combination of four bases (A, C, T, G).  DNA sequencing determines the order of these bases in a target gene; the data created are known as sequence reads and these reads can be compared to a DNA sequence database to determine the species present.

Last but not least, the composition of proteins in fish differs between species. Using this knowledge, fish speciation analysis can be conducted by a protein profiling technique. Similar to the RFLP technique discussed above, this technique separates the individual proteins by weight and this fingerprint is then compared to a profile of known species.

The methods available are continually being improved, such as increasing the DNA sequence and RFLP databases.

In time, testing is becoming more affordable and a key example is Next Generation Sequencing. This started at a cost of approximately $5,000 per megabase of DNA in September 2001 to $0.014 in October 2015 ³​. The rapid development and reduction in costs of molecular biology methods will in the future provide customers with a rapid and highly specific method.

The horsemeat scandal has enabled food testing laboratories to focus on the development of methods using molecular techniques not only in the authenticity testing sector but also allergens and rapid pathogen detection.

References​​

1. Oceana Study Reveals Seafood Fraud Nationwide n.d, accessed on 29/01/2016, http://oceana.org/sites/default/files/National_Seafood_Fraud_Testing_Results_Highlights_FINAL.pdf​
2. Too cheap to be true: Seafood fraud in Brussels, November 2015,  accessed on 29/01/2016, https://eu.oceana.org/sites/default/files/421/oceana_factsheet_seafood_fraud_brussels_eng.pdf​
3. Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP), January 15 2016, accessed on 11/02/2016,  www.genome.gov/sequencingcosts​

• Dr Emma Ridley is business manager (Molecular Biology) – Food & Waters UK at ALcontrol Laboratories. Her expertise covers ALcontrol's services including meat speciation, fish identification, GMO screening, rapid microbiological methods and allergen testing. She has previously worked in laboratories at Cornell University, Oxitec and Syngenta. Dr Ridley has worked with molecular techniques, including DNA microarray analysis, 454 pyrosequencing, end-point PCR and real-time PCR.