A multiplex real-time PCR screening assay for routine species identification of four commercially relevant crustaceans
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.