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

タグ

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記事を引用
Nested-PCR to Detect a Specific Viral Genomic Sequence. J. Vis. Exp. (Pending Publication), e21359, doi: (2023).

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