Low-cost fish species are often used to adulterate or substitute for Atlantic salmon products, posing a serious threat to market order and public health. Hence, reliable techniques are urgently needed to detect Atlantic salmon adulteration. In this study, a precise method for identifying and quantifying adulterated Atlantic salmon with rainbow trout based on droplet digital PCR (ddPCR) testing was developed. Species-specific primers and probes were designed targeting the single-copy nuclear gene myoglobin of two salmonids. A quantitative formula for calculating the mass fraction of adulterated Atlantic salmon with rainbow trout was established based on a one-step conversion strategy, in which the DNA copy number ratios were directly transformed to meat mass fractions by introducing a fixed constant (the transfer coefficient). The dynamic range of the established ddPCR method was from 1% to 90%, with a limit of detection (LOD) of 0.2% and a limit of quantification (LOQ) of 0.8% for rainbow trout in Atlantic salmon, respectively. The quantification method demonstrated an acceptable level of repeatability and reproducibility, as the values of the relative standard deviation (RSD) for the tested meat mixtures with the known fractions were all less than 5%. Thermal and freezing treatments, as well as adding food additives within the recommended dosage limits, had no significant effect on the quantification accuracy. The method was successfully applied to detect rainbow trout adulteration in commercial raw and processed Atlantic salmon products. In comparison to real-time quantitative PCR (qPCR) testing, the established ddPCR method exhibited a higher level of stability and accuracy. Overall, the ddPCR-based quantitative method exhibited high levels of accuracy, stability, sensitivity, and practicability, suitable for applications in the routine surveillance and quality assurance of salmon products.
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