This paper details the within-laboratory and inter-laboratory trial validation of a multiplex real-time PCR method for the simultaneous, sensitive and specific detection and semi-quantitative estimation of the nut species - peanut, hazelnut, walnut and cashew in processed food. The assay developed for the 4 species of nuts (peanut, hazelnut, walnut and cashew) was based on a TaqMan™ real-time PCR method, which targeted  multicopy sequences from mitochondrial, ribosomal RNA genes and chloroplasts, respectively. A series of prepared cookies, sausages, sauce powders, and veggie burgers spiked with different amounts of the 4 defatted nuts were used for the validation trials.The within-laboratory trial checked the specificity, crosstalk, sensitivity [limit of detection (LOD) including asymmetric LOD], precision and trueness of the assay. The inter-laboratory trial with 12 participating laboratories conducted both qualitative and quantitative determinations, and determined trueness/recoveries, precision, and measurement uncertainty. Using multicopy target sequences, a very sensitive detection of the allergenic ingredients is possible. Within the collaborative trial, a concentration of 0.64 mg/kg (i.e. approx. 0.1–0.2 mg “nut” protein/kg) could be reliably detected in a processed cookie matrix. With regards to quantitative analysis, there was insufficient recovery data (bias) resulting in measurement uncertainties of more than 50%. The results of in-house tests suggest that roasting of nuts is the main factor inducing deviant (low) recoveries.


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