Nested-PCR to Detect a Specific Viral Genomic Sequence

Overview

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 (challenge virus standard-11) and stored at -80°C. The negative control contains RNase-free ddH₂O.
4. Mix the contents of the RT tubes by vertexing; then, centrifuge briefly.
5. 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

1. Keep the PCR reagents listed in Table 2 on ice in a clean room until use; then, thaw and vortex them.
2. Prepare the first-round PCR mix in a 0.2 mL PCR tube with the reagents listed in Table 2.
3. 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.
4. Transfer the sealed tubes to a PCR thermal cycler and cycle using the parameters listed in Table 3.

3. Second-round PCR

1. Prepare the second-round PCR mix in a 0.2 mL PCR tube using the reagents listed in Table 4.
2. 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.
3. 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

Materials

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