Development and Validation of a Fast DNA Extraction Protocol for Fish Products

Fish species mislabeling is a typical form of food fraud widely reported around the world. The most applied technique in the detection of fish species mislabeling is based on DNA and the primary step of most DNA-based methods is to obtain sufficient amount of high-quality DNA quickly and efficiently. However, traditional DNA extraction method, for instance the salting-out method, can be greatly compromised by the fundamental step of cell lysis that generally costs hours or even overnight. Boiling protocol (BP), which can be achieved by an incubation for 10-15 min at 95-100 degrees C in a heat block or boiling water bath, is one of the simplest protocols for cell lysis. In this study, we applied BP method, together with a modified lysate, for DNA extraction from simulated fresh, frozen, and roasted fish products. Traditional proteinase K-based lysis method (TP) was used as the control and compared with BP method in DNA concentration, purity, conventional PCR, and real time PCR. Finally, BP method was validated on commercial fish products, followed by DNA barcoding to identify the labeling accuracy. The results highlighted a lower concentration and A(260)/A(230) ratio for DNA extracted by BP method than TP method for most species, regardless of the simulation methods. While conventional PCR amplification of 650 bp COI barcode can be achieved for all species in all simulations. Moreover, no significant difference (p > 0.05) was observed with Ct value for the same species between BP method and TP method. Total DNA can be successfully extracted with BP method for all commercial fish products and poor labeling accuracy was revealed for commercial roasted fish fillet products, 72.7% of which were identified as containing multiple species. Therefore, in light of the lower demand for time and cost, BP method can be considered as a valid alternative of TP method.