Optimization of a magnetic bead-based DNA extraction method combined with 12S rRNA barcoding for species traceability in fish oil products

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. • Development of a highly efficient magnetic bead-based DNA extraction method for fish oil: Due to severe DNA fragmentation during processing and the presence of high lipid content and antioxidants that inhibit PCR amplification, conventional DNA extraction methods often fail to yield DNA of sufficient quality for molecular identification. • Commercial kits also perform suboptimally, limiting the application of DNA barcoding in fish oil authentication. • To address this, we innovatively introduced a precisely quantified linear DNA fragment as an internal reference to accurately evaluate extraction efficiency. • Based on this, we systematically optimized key parameters including magnetic bead surface functional groups, particle size, quantity, binding buffer composition, washing conditions, and elution parameters. • The established protocol demonstrated significantly higher DNA yield and purity compared to several commercial kits ( p < 0.05). • Accurate species identification in fish oil using 12S rRNA barcoding: Due to the trace amounts of DNA in fish oil, direct sequencing is often unfeasible. • We developed a targeted amplification and sequencing system based on the 12S rRNA barcode region. • Using this system, we successfully recovered and sequenced 12S rRNA amplicons from 10 commercial fish oil products. • The results identified several instances of mislabeling, where the actual species did not match the label declarations, highlighting the substitution of high-value fish species with low-cost alternatives.