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.