Source: Marston, D. A. et al. Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis. J. Vis. Exp. (2019).
This video demonstrates a quantitative method for detecting viral infection using real-time reverse transcription PCR. The reverse transcription step converts the RNA to cDNA, which is then amplified via PCR cycles. The presence of viral RNA in the sample is confirmed by analyzing the amplification and dissociation curves of DNA obtained from the PCR.
1. Quantification of RNA Using a Micro-volume Spectrophotometer
2. Preparation of RNA Dilution Series to Determine End Point Sensitivity
3. Preparation of Real-time RT-PCR Reactions
Table 1: Pan-lyssavirus real-time RT-PCR master mix reagents.
Reagent | μL/Reaction |
Molecular-grade water | 7.55 |
2x Universal RT PCR reaction mix | 10 |
Primer Forward [20 μM] | 0.6 |
Primer Reverse [20 μM] | 0.6 |
RT enzyme mix | 0.25 |
Total per reaction | 19 |
Table 2: Pan-lyssavirus real-time RT-PCR primer details.
Assay | Primer name | Primer role | Sequence 5'-3' | Posição1 |
Lyssavirus | JW12 | RT-PCR | ATG TAA CAC CYC TAC AAT G | 53-73 |
N165 | PCR | GCA GGG TAY TTR TAC TCA TA | 165-146 | |
ß-actin | ß-actin intronic | PCR | CGA TGA AGA TCA AGA TCA TTG | 1051-1072 |
ß-actin reverse | RT-PCR | AAG CAT TTG CGG TGG AC | 1204-1188 | |
Primer positions are given in relation to Pasteur virus sequence (M13215) and mouse ß-actin gene sequence (NM_007393) |
Table 3: Pan-lyssavirus real-time RT-PCR cycling conditions.
Stage | Cycles | Temperature | Time | Data Collection |
Reverse Transcription | 1 | 50 °C | 10 min | |
RT inactivation/initial denaturation | 1 | 95 °C | 5 min | |
Amplification | 40 | 95 °C | 10 s | |
60 °C | 30 s | endpoint | ||
Dissociation curve analysis | 1 | 9 °C | 1 min | |
55 °C | 1 min | |||
55 – 95 °C | 10 s | all points |
The authors have nothing to disclose.
Art Barrier pipette tips (various sizes) | Thermofisher | various | |
Centrifuge | Beckman | Allegra 21R | Rotor capable of holding 96 well plates required. Step 3.8. |
Centrifuge (micro) | Sigma | ||
Finnpipettes (to dispense 0.5-1000 µL) | Thermofisher | various | |
iTaq Universal SYBR Green One-Step RT-PCR kit | Bio-Rad | 172-5150 | Equivalent kits can be used if validated |
MX3000P or MX3005P real-time PCR system | Stratagene | N/A | Equivalent machines can be used if validated |
MicroAmp reaction plate base | Any suitable | Used to hold tube strip and plates securely. | |
Optically clear flat clear strips (8) | ABgene | AB-0866 | |
Perfect fit frame (if using tube strips) | Stratagene | N/A | Specific to machine |
Primers: for primer details see Table 2. | Ordered at 0.05 µmole scale HPLC purified. | ||
Thermo-Fast 96-well plates, non-skirted | ABgene | AB-600 | |
Thermo-Fast strips (8) Thermo-tubes | ABgene | AB-0452 | |
Vortex machine / Whirlimixer | Fisons Scientific equipment | SGP-202-010J | |
Unless stated, alternative equipment can be used |