Abstract: In order to establish a rapid method for the identification of adulterated fish and fish products, we designed three pairs of species-specific primers targeting the mitochondrial displacement-loop (D-loop) region of bluefin tuna (Thunnus thynnus), the mitochondrial 16S rRNA gene of sablefish (Anoplopoma fimbria) and the mitochondrial ND4L gene of oilfish (Lepidocybium flavobrunneum), respectively, yielding amplicons having significantly different melting temperatures (Tm). A multiplex real-time polymerase chain reaction (real-time PCR) combined with melting curve analysis method was established for the rapid identification of bluefin tuna-derived, sablefish-derived and oilfish-derived ingredients in fish and fish products, and it was evaluated in terms of specificity and sensitivity and then applied in the detection of commercial meat products. The results suggest that the method had good specificity and sensitivity, and was able to detect 0.001 ng of DNA from single species and 0.1 ng of DNA from mixed species. Meanwhile, it was indicated that the relative detection limits of bluefin tuna, sablefish and oilfish were 0.5%, 1% and 0.5%, respectively. The method was successfully applied to commercially available samples.

Key words: fish; adulteration; multiplex real-time polymerase chain reaction; melting curve analysis; bluefin tuna; sablefish