Nested-PCR to Detect a Specific Viral Genomic Sequence
Published: May 31, 2023
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 …
開示
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 |
