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Adulteration or mislabelling of seafood products is a major issue and a problem prevalent worldwide. DNA sequencing-based methods currently used for species identification help to reveal adulteration, but drawbacks like their requirement for sophisticated equipment, which is not available in every food monitoring laboratory, or their time-consumption render most of them insufficient for routine analysis. This paper presents a TaqMan-probe based multiplex real-time PCR screening assay for identification of the four commercially relevant crustacean species Penaeus monodon , Litopenaeus vannamei , Pleoticus muelleri and Nephrops norvegicus. All newly developed primer/probe sets of the multiplex real-time PCR assay exhibited a limit of detection between 0.2 pg–2 pg DNA and amplification efficiencies in a range of 97.1–100.9%. The specificity of the assay was confirmed by testing more than 30 crustacean species demonstrating the great potential for a reliable species identification. In addition, the performance of the method was evaluated with varyingly processed crustaceans as well as with commonly used spices and herbs. Average Cq-values were determined for each tested species. Finally, all primer/probe sets were further investigated and optimised for use within a multiplex PCR to offer a rapid authenticity verification of crustacean seafood products. • Development of a multiplex real-time PCR for the screening of relevant crustaceans. • Assay targets P. monodon , L. vannamei , P. muelleri and N. norvegicus. • Sensitivity, robustness and efficiency of the assay meet performance criteria. • Assay specificity demonstrated by analysis of over 30 crustacean species. [ABSTRACT FROM AUTHOR]

Seafood products are particularly vulnerable to food fraud and mislabelling, which may have negative implications on fisheries resources, economy, consumer health and trust. This is a considered problem for scallops, since these high-valued seafood products are usually sold without their morphologically characteristic shells. Scallop products differ in taste and value and usually species of the genus Pecten spp. are especially expensive in many European countries. The aim of the present study was the development of a multiplex TaqMan real-time PCR assay that allows a rapid and reliable authentication of the three commercially important species/genera Pecten spp. (usually King scallop P. maximus), Atlantic sea scallop Placopecten magellanicus , and Japanese scallop Mizuhopecten yessoensis. The design of primers and probes was based on mitochondrial 16S rRNA gene amplifying fragments of 138–198 bp. Following the optimization of the multiplex real-time PCR assay, analyses on efficiency, limit of detection, specificity, robustness, and crosstalk were conducted for validation purpose. Average Cq values of 20 ng DNA obtained for Pecten spp. were 17.64 ± 0.89, for P. magellanicus 18.42 ± 0.83, and for M. yessoensis 17.08 ± 0.79. Non-target species produced either no fluorescence signal or the Cq differed significantly from those of target species (p < 0.01). Finally, the newly developed real-time PCR 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. The results emphasize the need of a control method that allows the rapid analysis of sufficient sample quantities. The study is one of the first which presents a multiplex TaqMan real-time PCR approach for the authentication of commercially important seafood species. • Development of a multiplex real-time method for the identification of scallops. • Assay targets Pecten spp., Mizuhopecten yessoensis , and Placopecten magellanicus. • High mislabelling of scallop products was found for samples from the German market. • Results underline the need for regular control and rapid authentication methods. [ABSTRACT FROM AUTHOR]