Interlaboratory study on real-time PCR detection and quantification of the European anglerfish, pike, and seabream parvalbumin gene

Abstract

AbstractThis study presents a large-scale interlaboratory comparison (ILC) aimed at detecting and quantifying DNA from two European anglerfish (Lophius budegassa, Lophius piscatorius), pike (Esox lucius) and sea bream (Spondyliosoma cantharus) using real-time qPCR. To detect amplification of the parvalbumin genetic marker, single and multiplex qPCR assays using EvaGreen® dye or TaqMan™ probes were used. Genomic DNA isolated from target fish species and an advanced DNA calibrator, gBlocks® gene fragments, were used as standards. The DNA of anglerfish, pike and sea bream as well as their mixtures were analysed together with 14 other non-target fish species. All target fish samples were correctly identified by the participating laboratories. Qualitative assessment of anglerfish and seabream DNA showed an accuracy rate of 100%, while pike DNA achieved a match rate of 99%. Validation of quantitative protocols in four different laboratories consistently achieved z-scores below 2, indicating satisfactory performance and confirming the high degree of similarity of laboratory results. Furthermore, high accuracy and efficiency were demonstrated for the quantification of anglerfish and seabream DNA by triplex qPCR using TaqMan™ probes. Regarding the selected gene marker, the major fish allergenic protein parvalbumin enables indirect detection and quantification of the allergen in the sample. Therefore, the use of proposed protocols can significantly contribute to protecting the health of consumers and to controlling the food market. Graphical abstract