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.