qPCR experiment design
Quantitative Real Time PCR (qPCR) assays were performed with AB 7500 (Applied Biosystem) to test the three primer pairs. First, for each primer pair, M. monachus tissue samples were used to set amplification efficiency (E) and limit of detection (LOD) and limit of quantification (LOQ), according to Bustin et al ., 2009 and Klymuset al ., 2019. Ten-fold serial dilutions were used to generate the standard curve.
All 73 samples plus no-template negative controls were run in triplicate, using the following qPCR conditions: an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing-elongation at 56 °C for 1 min. A final dissociation stage was performed. Amplification reaction consisted of 5.0 μl SsoFast EvaGreen Supermix with Low ROX (Bio-Rad), 0.1 μl each [10 μM] primer solution, 2 μl DNA sample, and 2.8 μl of Milli-Q water.
Ct (Threshold Cycles) values were converted into counts (DNA copies) according to Bruno et al ., 2017. When qPCR copy number outputs were below the limit of quantification (LOQ), but above the theoretical limit of qPCR (three copies per reaction according to Bustin et al . (2009)), they were addressed as ‘detectable but not quantifiable’ (DBNQ). For all the reactions, primer specificity was verified by the calculated TM.