Development of sensitive and specific real-time PCR systems for the detection of crustaceans in food

Abstract

Crustaceans are known allergens for a remarkable number of people. For the detection of traces of crustaceans in food, a specific and sensitive real-time PCR method was developed. An approximately 205 bp long fragment of the mitochondrial 16S rRNA gene was chosen as molecular target region for the detection systems. The DNA sequence of this fragment was determined from 13 species belonging to different families and checked for homologies. Based on these data, primer–probe systems were developed for the economically relevant decapods within the class of Malacostraca belonging to the families Penaeidae, Palinuroidea, Astacoidea, Nephropoidea, Cancridae and Caridea. The specificity of the primer–probe systems was checked for inclusivity using DNA extracted from 17 different crustaceans. Exclusivity tests were carried out by analysing DNA samples derived from 21 mammals, six birds, 13 fishes, two molluscs and two insect species. Except for two systems, the molecular detection systems were optimised to be highly specific for the crustaceans. False positive signal was produced by DNA extracted from the hoverfly (Psilota rubra) in the system targeting the family Astacoidea and the common green lacewing (Chrysoperla carnea) in the systems targeting the family Astacoidea and Cancroidea. The LOD95% was close to the theoretical value of 2.96 copies per reaction. The sensitivity of the real-time PCR systems was determined using dilution series of crustacean DNA in rainbow trout DNA as animal matrix, and by artificial contamination of fish sticks and by artificial contamination of cassava chips with crustacean meat. The sensitivity ranging from 10 to 0.01 ppm is considered being appropriate for food analysis.