Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European Bat Lyssavirus Type 1
Table 1
Characteristics of the commercial master mix kits used. In (a) the reactions mix and thermocycling conditions for the five RT kits are detailed, in (b) the reactions mix and thermocycling conditions for the five qPCR SybrGreen kits (two-step), and in (c) the reactions Mix and thermocycling conditions for the five qRT-PCR SybrGreen kits (one-step) used in this study.
(a) Reactions mix and thermocycling conditions for the five RT kits
Manufacturer
Reverse transcription kit
RTase and primers
Thermocycling conditions
Reaction mix**
Bio-Rad
Iscript select cDNA synthesis kit
M-MLV RT* Random primer
5 min-25°C 30 min-42°C 5 min-85°C
4 L Iscript Mix (5x) + 2 L Random primer + 1 L Iscript RTase + 8 L RNase free water
Qiagen
QuantiTect Reverse Transcription Kit
Quantiscript RTase Mix OligodT + random primer
2 min-42°C 30 min-42°C 3 min-95°C
2 L gDNA Wipeout Buffer (7x) + 4 L Quantiscript RT Buffer + 1 L Quantiscript RTase + 1 L RT primer + 7 L RNase free water
Fermentas
Maxima H Minus First Strand cDNA Synthesis Kit
M-MLV RT* Random primer
5 min-65°C 10 min-25°C 30 min-50°C 5 min-85°C
4 L RT Buffer (5x) + 1 L Random primer + 1 L dNTP (10 mM) + 1 L Maxima H Minus Ez Mix + 8 L RNase free water
Invitrogen
SuperScript Vilo Master Mix
M-MLV RT* Random primer
10 min-25°C 30 min-42°C 5 min-85°C
4 L SuperScript Vilo MasterMix (5x) + 11 L RNase free water
Quanta
qScript cDNA Synthesis Kit
M-MLV RT* Mix OligodT + random primer
5 min-22°C 30 min-42°C 5 min-85°C
4 L qScript Reaction Mix (5x) + 1 L qScript RTase + 10 L RNase free water
Moloney Murine Leukemia Virus. **Total volume of reaction is 20 L containing 5 L of RNA sample and 15 L of reaction mix. All RT assays were performed on the Mastercycler Eppendorf thermal cycler. RTase: reverse transcriptase.
(b) Reactions mix and thermocycling conditions for the five qPCR SybrGreen kits (two-step)
Manufacturer
qPCR SybrGreen kit
Thermocycling conditions
Reaction mix*
Bio-Rad
SsoFast EvaGreen Supermix
30 sec-95°C 45 cycles of 30 sec-95°C; 30 sec-55°C; 30 sec-72°C Melt Curve (60°C to 95°C)
Total volume of 20 L containing 2 L of cDNA and 18 L of a reaction mix (1x Sso Fast master mix, 0.35 M [0.5 M] each of pan-Lyssavirus primers)
Qiagen
QuantiTect SYBR Green PCR Kit
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)
Total volume of 25 L containing 2 L of cDNA and 23 L of reaction mix (1x Quantitect Master mix, 0.5 M [0.6 M] each of pan-Lyssavirus primers)
Fermentas
Maxima SYBR Green qPCR Master Mix
10 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)
Total volume of 25 L containing 2 L of cDNA and 23 L of reaction mix (1x Maxima SYBR Green master mix, 0.4 M [1 M] each of pan-Lyssavirus primers)
Invitrogen
Express SYBR GreenER qPCR Supermix Universal
2 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)
Total volume of 20 L containing 2 L of cDNA and 18 L of reaction mix (1x Express SYBR GreenER Supermix, 0.4 M [2 M] each of pan-Lyssavirus primers) [50 nM of ROX dye]
Quanta
Perfecta SYBR Green SuperMix
3 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)
Total volume of 25 L containing 2 L of cDNA and 23 L of reaction mix (1x Perfecta SYBR Green Supermix, 0.3 M [1 M] each of pan-Lyssavirus primers)
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 [ ].
(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 M [2 M] 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 M [2 M] 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 M [0.3 M] 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 M [0.5 M] 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 M [0.2 M] each of pan-Lyssavirus primers
Total volume of reaction is 25 L containing 2 L of RNA sample and 23 L 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 [ ].
