multiplex real-time pcr (2)


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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7932204055?profile=RESIZE_400x Scallops are high value seafood products usually sold without their characteristic shells. Each species differs in its taste and value with the Pecten spp.  scallops attracting higher prices in Europe. German researchers have developed a multiplex real-time PCR method to reliably identify the main commercial scallop species: Pecten spp. (usually King scallop P. maximus), Mizuhopecten yessoensis (Japanese scallop), and Placopecten magellanicus (Atlantic sea scallop). Primers and probes  based on mitochondrial 16S rRNA gene amplifying fragments of 138–198 bp were used, and non-targeted species gave either no fluorescent signal or cycle numbers (Cq) very different from the targeted species. The newly developed assay was tested on commercial samples from German supermarkets and fishmongers accompanied by simultaneous verification through Sanger sequencing, which revealed a high mislabelling rate of 48%, especially for products purchased at fishmongers. 

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