Nested-PCR to Detect a Specific Viral Genomic Sequence

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

1. Remove the RT reagents listed in Table 1 from the freezer, keep them on ice, and thaw and vortex them before use.
2. 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.
3. 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 (ch....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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