(c) Reactions mix and thermocycling conditions for the five qRT-PCR SYBR Green kits (one-step)
| Manufacturer | qRT-PCR SybrGreen kit |
Thermocycling conditions | Reaction mix* |
|---|---|---|---|
| Qiagen | QuantiTect SYBR Green RT-PCR Kit | 30 min-50°C, 15 min-95°C 45 cycles of 30 sec-95°C; 30 sec-55°C; 30 sec-72°C Melt Curve (60°C to 95°C) |
1x QuantiTect SYBR Green master mix, 1% Quantitect RT mix, 0.6 µM [2 µM] each of pan-Lyssavirus primers |
|
| |||
| Qiagen | Rotor Gene-1 step: Rotor-Gene SYBR Green RT-PCR Kit | 30 min-50°C, 5 min-95°C 45 cycles of 30 sec-95°C; 30 sec-55°C; 30 sec-72°C Melt Curve (60°C to 95°C) |
1x Rotor-Gene master mix, 1% Rotor-Gene RT mix, 1 µM [2 µM] each of pan-Lyssavirus primers |
|
| |||
| Fermentas | Verso 1-step QRT-PCR Kit | 30 min-50°C, 15 min-95°C 45 cycles of 30 sec-95°C; 30 sec-55°C; 30 sec-72°C Melt Curve (60°C to 95°C) |
1x Verso SYBR Green master mix, 5% RT enhancer, 1% Verso enzyme mix, 0.2 µM [0.3 µM] each of pan-Lyssavirus primers |
|
| |||
| Invitrogen | SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit | 10 min-50°C, 5 min-95°C 45 cycles of 30 sec-95°C; 30 sec-55°C; 30 sec-72°C Melt Curve (60°C to 95°C) |
1x SuperScript III Platinium master mix, 2% enzyme mix, 0.3 µM [0.5 µM] each of pan-Lyssavirus primers |
|
| |||
| Quanta | qScript One-Step SYBR Green RT-qPCR Kit | 30 min-50°C, 5 min-95°C 45 cycles of 30 sec-95°C; 30 sec-55°C; 30 sec-72°C Melt Curve (60°C to 95°C) |
1x qScript master mix, 2% qScript enzyme mix, 0.4 µM [0.2 µM] each of pan-Lyssavirus primers |
*Total volume of reaction is 25 µL containing 2 µL of RNA sample and 23 µL of reaction mix. The PCR reaction protocol corresponds to PCR assays performed on the Rotor Gene Q MDx. The concentrations of primers used on the real time thermocycler Mx3005P are shown in brackets [ ].