Table 3. Optimisation experiments performed for the three best performing RT-qPCR systems*.
| Factor | Mastermix | Sample volume | Reaction volume | Annealing temperature# | Primer concentration | Probe concentration |
|---|---|---|---|---|---|---|
| I Annealing temperature | Superscript-III | 5μl | 25μl | 54, 56, 58, 60, 62 and 64°C | 400nM | 200nM |
| II Primer concentration | Superscript-III | 5μl | 25μl | 1. 60°C 2. 62°C 3. 56°C |
200, 300, 400, 500, 600 and 800 nM | 200nM |
| III Probe concentration | Superscript-III | 5μl | 25μl | 1. 60°C 2. 62°C 3. 56°C |
600nM | 100, 200, 300 and 400nM |
* All experiments were performed with G1-1326 tenfold dilutions.
#Annealing temperature for the Pyke, NS2A and NS3 systems were 60, 62 and 56°C respectively.