Abstract: Objective: The aim of this study was to establish a new method for the rapid and accurate quantification of walnut-derived ingredients and adulterated ingredients, mainly derived from soybean, in commercial walnut beverage using droplet digital polymerase chain reaction (ddPCR). Methods: This method was based on the ratio between the numbers of target gene copies per unit mass of walnut and soybean, which could represent the relationship between gene copy number and the mass of plant materials. The soybean lectin gene and the walnut Jugr2 gene were chosen as the target genes according to the commercial inspection standard SN/T 1961-2013. The specificity of the assay was evaluated by testing DNA from six different species using the species-specific primers and probes. The results showed that there was no cross-reaction among the target species and high sensitivity was observed. The ddPCR assay showed a limit of detection (LOD) for added soybean-derived ingredients in walnut protein drink was 0.5% with a relative error of 5.6%. The ratios of target gene copy numbers between soybean and walnut in pure samples of five different masses and between the two species in five mixtures with different proportions were determined three times, and the experimental data were fitted to a linear equation to calculate the ratio between gene copy numbers per unit mass soybean and walnut (Csoybean/Cwalnut ratio = 1.771 1). Furthermore, we applied this method to test 11 commercial brands of walnut beverage, and it turned out that walnut-derived ingredients were detected in all these samples and 6 of them were found to also contain soybean-derived ingredients, out of which 4 were adulterated with more than 10% soybean and the others contained as low as 0.2% soybean, which may be unintentionally incorporated during the production process. Conclusion: The ddPCR assay can provide a rapid and accurate quantitative method for the identification of adulterated walnut beverage.

Key words: walnut protein drink, duplex digital droplet polymerase chain reaction (PCR), mass ratio, adulteration