Development and Validation of a Fast DNA Extraction Protocol for Fish Products
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Development and Validation of a Fast DNA Extraction Protocol for Fish Products Xiong Xiong 1
&
Manhong Huang 1 & Fangying Yuan 1 & Lixia Lu 1 & Xiaohui Xiong 1
Received: 13 November 2018 / Accepted: 28 May 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract 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 °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 A260/A230 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. Keywords DNA extraction . DNA barcoding . Fish products . Boiling protocol
Introduction The explosive growth of scientific literatures related to fish traceability over the last years has revealed a global issue of fish species adulteration (Giusti et al. 2018; Hellberg et al. 2019; Horreo et al. 2019; Hu et al. 2018; Sultana et al. 2018; Zeng et al. 2018). Financial incentive is widely recognized as the greatest motivation for this illicit conduct, and an 11% rate of mislabeling low-value Atlantic salmon as high-value Pacific salmon has caused an illegal profit of seven million
Xiong Xiong and Manhong Huang contributed equally to this work. * Xiong Xiong [email protected] * Xiaohui Xiong [email protected] 1
College of Food Science and Light Industry, Nanjing Tech University, No. 30 Puzhu Road South, Nanjing 211800, China
dollars in America alone (Cline 2012; Jacquet and Pauly 2
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