Asadatun Abdullah

Authentication of raw and processed tuna from Indonesian markets using DNA barcoding, nuclear gene and character-based approach

Abstract Establishing seafood authentication methods is an important task for fisheries research laboratories and food control authorities. Nowadays, the extent of fish species substitution is suspected being greater than ever before in commercial markets. In order to provide reliable polymerase chain reaction (PCR)-based authentication systems for tunas, we collected and analyzed authentic tuna reference samples and tuna-food products from Indonesian markets. Our analytical methods mainly relied on identification using the mitochondrial cytochrome c oxidase subunit I (COI) gene, as a genetic marker for “DNA barcoding,” as well as the rhodopsin (RH1) gene as a nuclear marker. Additionally, we identified species-specific nucleotide diagnostic positions (characters) to complete the results obtained basic local alignment search (BLAST) and phylogenetic analysis. Authentication results of tuna-food products showed relatively successful amplification for the COI gene; RH1 acted as an alternative solution for some of the samples, which had failed to react in COI-PCR. Species of the genus Thunnus could not be unambiguously differentiated by BLAST and phylogenetic analysis (neighbor-joining tree) in all cases due to the high similarity of the COI sequences. However, the character-based identification method was found to be helpful for species assignment in case of tuna-food products. Therefore, our findings demonstrated that the COI gene could be more reliable used as a tool for Indonesian commercial tuna products authentication, if the sequencing results were combined with the character-based identification using differences at certain nucleotide positions.

The differentiation of tuna (family: Scombridae) products through the PCR-based analysis of the cytochrome b gene and parvalbumin introns.

BACKGROUND In spite of the many studies performed over the years, there are still problems in the authentication of closely related tuna species, not only for canned fish but also for raw products. With the aim of providing screening methods to identify different tuna species and related scombrids, segments of mitochondrial cytochrome b (cyt b) and nuclear parvalbumin genes were amplified and sequenced or subjected to single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) analyses. RESULTS The nucleotide diagnostic sites in the cyt b gene of five tuna species from Indonesia were determined in this study and used to construct a phylogenetic tree. In addition, the suitability of the nuclear gene that encodes parvalbumin for the differentiation of tuna species was determined by SSCP and RFLP analyses of an intron segment. RFLP differentiated Thunnus albacares and from T. obesus, and fish species in the Thunnus genus could be distinguished from bullet tuna (Auxis rochei) by SSCP. CONCLUSIONS Parvalbumin-based polymerase chain reaction systems could serve as an additional tool in the detection and identification of tuna and other Scombridae fish species for routine seafood control. This reaction can be performed in addition to the cyt b analysis as previously described.

DNA barcoding for the species identification of commercially important fishery products in Indonesian markets

Summary The DNA barcoding approach was used for the species identification of 44 Indonesian commercial fishery products. Additionally, the intronless nuclear rhodopsin gene fragment (RH1) was added to the analysis to enable the identification of species not yet barcoded and possible hybrids. The 655-bp cytochrome C oxidase subunit I (COI) gene fragment marker was successfully amplified and used to identify 86% of the total fish samples at the species level using the BOLD and BLAST public databases. Moreover, the RH1 marker was used to complete COI analysis. For a number of fish species, the COI sequences (six species) and RH1 sequences (eight species) were the first entries submitted to GenBank. This study demonstrated that COI barcoding is a promising tool for Indonesian fishery products and confirmed that it could be adopted in the future for regular seafood control as part of the Indonesian integrated food traceability system.

Authentication of closely related scombrid, catfish and tilapia species by PCR-based analysis and isoelectric focusing of parvalbumin

In recent decades, the authentication of fishery products has relied mainly on DNA analysis of mitochondrial genes; however, these methods cannot distinguish hybrids from their respective maternal species. As an alternative for wild or farmed hybrid fish authentication, we developed assays based on the amplification of a parvalbumin intron by exon-primed intron-crossing PCR. Parvalbumins, the major class of fish allergens, are encoded by nuclear genes and are present in high concentrations in the light muscle of many fish species. Amplicons of analysed fish species were characterized by sequencing (tunas), single strand conformation polymorphism (SSCP) (scombrid, catfish, tilapia, and snapper species) and restriction fragment length polymorphism (RFLP) (catfish) analyses. The SSCP method differentiated catfish, tilapia, snapper and scombrid species except tunas. Tunas of the genus Thunnus had an unexpected low variability of intron sequences, which prevented their differentiation by sequencing or SSCP. For study of hybrid catfish, RFLP analysis with Ban I endonuclease was used to construct specific DNA fragment profiles. Isoelectric focusing (IEF) of sarcoplasmic proteins was a rapid screening method to identify catfish, tilapia and snapper species because of their specific protein patterns. The heat-stable, anodic protein bands of these patterns presumably belong to parvalbumins, the major class of fish allergens. PCR and IEF techniques for analysing parvalbumins can be used as routine methods to control the labelling of fish products with the exception of tuna products.