Fig. 5

Resolution of the assay. a Amplification curves obtained with control primer pair 1 (dashed lines) or control primer pair 2 (solid lines) and different copy numbers of both templates present in equimolar ratio shown as means of triplicates performed in independent experiments with similar results (n = 5). The corresponding standard curves are shown on the right. Correlation coefficient (R ²), slope (M), y-intercept (B) and amplification efficiencies (E) calculated with the equation E = 10^−1/M are indicated. b Copies obtained in different reactions (x) with control primer pair 1 or control primer pair 2 at different template concentrations were calculated using the equation: \({\text{copies}} = \left[ {{{{\text{N}}_{0} \left( {\text{X}} \right)} \mathord{\left/ {\vphantom {{{\text{N}}_{0} \left( {\text{X}} \right)} {{\text{N}}_{0} \left( {\text{con2}} \right)}}} \right. \kern-0pt} {{\text{N}}_{0} \left( {\text{con2}} \right)}}} \right] \cdot 10^{8} = 2^{{{{{\text{Cqz}} \cdot { \log }\left( {\text{Ez}} \right)} \mathord{\left/ {\vphantom {{{\text{Cqz}} \cdot { \log }\left( {\text{Ez}} \right)} {\log \left( 2 \right)\text{ - }{{{\text{Cqx}} \cdot { \log }\left( {\text{Ex}} \right)} \mathord{\left/ {\vphantom {{{\text{Cqx}} \cdot { \log }\left( {\text{Ex}} \right)} {\log \left( 2 \right)}}} \right. \kern-0pt} {\log \left( 2 \right)}}}}} \right. \kern-0pt} {\log \left( 2 \right)\text{ - }{{{\text{Cqx}} \cdot { \log }\left( {\text{Ex}} \right)} \mathord{\left/ {\vphantom {{{\text{Cqx}} \cdot { \log }\left( {\text{Ex}} \right)} {\log \left( 2 \right)}}} \right. \kern-0pt} {\log \left( 2 \right)}}}}}} \cdot 10^{8}\) with Cqz and Ez = Cq and E values obtained with control primer pair 2 and 10⁸ introduced copies. The efficiencies introduced into the equation were obtained either from the comparative quantitation tool of the Rotorgene software (red columns) or the standard curves shown in A (blue columns). Dark columns represent reactions using control primer pair 2, light columns control primer 1. Mean values ± SE of five independent experiments each performed in triplicate are presented