SOP-019 qPCR assay for the quantification of cod (Gadus morhua) environmental DNA

This protocol describes the specific determination and quantification of cod (Gadus morhua) from environmental DNA (seawater and sediment samples). Developed by the Thünen Institute of Fisheries Ecology, this assay is used for biomass analysis in the quantitative part of the FishGenome Project using real time quantitative Polymerase Chain Reaction (qPCR) technology. SCOPE & LIMITATIONS Analysis of eDNA from cod (Gadus morhua) sampled from water and sediments of the North Sea. For reliable and reproductive quantification, the Limits of Blank (LoB), Detection (LoD) and Quantification (LoQ) have been determined according to Armbruster and Pry (2008) and Bustin et al. (2009)(for further details on the calculation of these values see -section 9-): LoB = 11,6 copies per reaction, equivalent to Ct 36.0 LoD = 10 copies per reaction, equivalent to Ct 36.3 (as LoD < LoB, the LoB is used as LoD) LoQ = 16.40 copies per reaction, equivalent to Ct value 35.4 This method is valid when a copy number equivalent to or greater than 17 copies/reaction is available in the eDNA samples. In other words, any positive signal below the Ct (threshold cycle) value of 35.4 is considered negative. This is due to the high risk of false positives after Ct = 36. This protocol is working perfectly only with the polymerase “Applied Biosystems™, TaqMan™ Environmental Master Mix 2.0” (FisherScientific, Schwerte, Germany), the change of polymerase does not guarantee the performance and efficiency of the method described in this SOP.