Optimization of a magnetic bead-based DNA extraction method combined with 12S rRNA barcoding for species traceability...

Fish oil is a widely consumed nutritional supplement rich in ω-3 polyunsaturated fatty acids (PUFAs) and of high economic value, but it is susceptible to economically motivated adulteration, including the incorporation of lower-value fish species. Reliable methods for species authentication are therefore needed. In this study, we established and systematically optimized a magnetic bead-based DNA extraction protocol tailored for fish oil matrices. The method incorporates an exogenous DNA fragment as an internal control to quantitatively assess extraction efficiency, and key parameters-including bead surface chemistry, particle size and dosage, binding buffer composition, wash conditions, and elution temperature and duration-were evaluated and optimized. The final optimized protocol (500 nm hydroxyl-functionalized beads, 3 mol/L guanidine thiocyanate binding system, 70% ethanol wash, and 56 °C elution) achieved significantly higher DNA yield and purity than multiple commercial extraction kits. When coupled with 12S rRNA metabarcoding, the method enabled the recovery of amplifiable DNA from all ten commercial fish oil products tested and allowed species-level identification. The results revealed discrepancies between labeled claims and actual biological composition in several products, including indications of substitution of high-value deep-sea fish with lower-value species. Taken together, this workflow provides a practical technical basis for species traceability and authenticity evaluation of fish oil, and it may be further applicable to quality monitoring in other high-lipid food matrices.