Abstract
Source: Feng, Y., et al. Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses. J. Vis. Exp. (2019).
This video describes nested polymerase chain reaction, a technique that consists of two sequential PCR amplification processes using two primer sets. The first set of primers is intended to anneal to sequences upstream of the second set, resulting in selective amplification of specific gene sequences. This PCR is more sensitive and specific than a normal PCR and is widely used as a detection technique for various diseases.
Protocol
1. Reverse Transcription of the Viral RNA
- Remove the RT reagents listed in Table 1 from the freezer, keep them on ice, and thaw and vortex them before use.
- Prepare 12 µL of RT reaction mix in a 0.2 mL PCR tube with the reagents listed in Table 1. Allow for pipetting variations by preparing a volume of the master mix at least one reaction size greater than required.
- Add 8 µL sample, positive control RNA, or negative control to the RT reaction mix within a PCR workstation in a template room. The RT positive control is RNA extracted from the cell culture infected with fixed RABV strain CVS-11 (challenge virus standard-11) and stored at -80°C. The negative control contains RNase-free ddH2O.
- Mix the contents of the RT tubes by vertexing; then, centrifuge briefly.
- Load the reaction tubes into a thermal cycler. Set up the cDNA synthesis program with the following conditions: 42°C for 90 min, 95°C for 5 min, and 4°C on hold. Set the reaction volume to 20 µL. Start the RT run.
2. First-round PCR
- Keep the PCR reagents listed in Table 2 on ice in a clean room until use; then, thaw and vortex them.
- Prepare the first-round PCR mix in a 0.2 mL PCR tube with the reagents listed in Table 2.
- Add a 2 µL sample of cDNA or plasmid into the first-round PCR mix within a PCR workstation in a template room. The PCR positive control is CVS-11 cDNA prepared as mentioned in step 1.3 for the above RT method. The PCR negative control is dd H2O.
- Transfer the sealed tubes to a PCR thermal cycler and cycle using the parameters listed in Table 3.
3. Second-round PCR
- Prepare the second-round PCR mix in a 0.2 mL PCR tube using the reagents listed in Table 4.
- Add 2 µL of the first-round PCR product into the second-round PCR mix. In addition, include dd H2O as a negative control of the second-round PCR.
- Perform PCR thermal cycling using the same parameters as given in step 2.4.
Table 1: Reagents of reverse transcription for cDNA synthesis.
| Component | Volume per reaction (µl) |
| dNTPs (2.5 mM ) | 4 |
| Random Primer (50 μM) | 1.5 |
| Oligo(dT) 15 (50 μM) | 0.5 |
| M-MLV buffer (5x) | 4 |
| M-MLV reverse transcriptase (200 IU/µL) | 1 |
| RNasin (40 IU/µL) | 1 |
| Total volume | 12 |
Table 2: Reagents of the first-round PCR.
| Component | Volume per reaction (µl) |
| dNTPs (10 mM) | 1 |
| Ex-Taq (5 U/μL) | 0.3 |
| Taq Buffer (10x) | 5 |
| N127 (20 μM) | 1 |
| N829 (20 μM) | 1 |
| dd H2O | 39.7 |
| Total volume | 48 |
Table 3: Cycling parameters of the first- and second-round PCR
| Temperature | Time | Cycle |
| 94 °C | 2 min | 1 |
| 94 °C | 30 s | 35 |
| 56 °C | 30 s | 35 |
| 72 °C | 40 s | 35 |
| 72 °C | 10 min | 1 |
| 4 °C | ∞ |
Table 4: Reagents of the second-round PCR.
| Component | Volume per reaction (µl) |
| dNTPs (10 mM) | 1 |
| Ex-Taq (5 U/μL) | 0.3 |
| Taq Buffer (10x) | 5 |
| N371F (20 μM) | 1 |
| N371R (20 μM) | 1 |
| dd H2O | 39.7 |
| Total volume | 48 |
Divulgaciones
The authors have nothing to disclose.
Materials
| ddH2O | Various | Various | |
| dNTPs (10 mM) | TakaRa | 4019 | |
| dNTPs (2.5 mM) | TakaRa | 4030 | |
| Ex-Taq (5 U/μL) | TakaRa | RR001 | |
| Microcentrifuge tubes | Various | Various | |
| M-MLV reverse transcriptase (200 IU/µL) | TakaRa | 2641A | |
| Oligo (dT)15 | TakaRa | 3805 | |
| PCR Machine | BIO-RAD | T100 | |
| PCR Tubes | Various | Various | |
| Pipettors | Various | Various | |
| Random Primer | TakaRa | 3801 | |
| RNase Inhibitor (40 IU/µL) | TakaRa | 2313A | |
| RNase-free ddH2O | TakaRa | 9102 | |
| Taq Buffer (10x) | TakaRa | 9152A | |
| Tips | Various | Various | |
| Vortex mixer | Various | Various |
