Aug 05, 2024
Version 1
SeV standard and copyback genomes PCR Protocols V.1
- Carolina Lopez1
- 1Washington University
External link: https://doi.org/10.1128/jvi.00894-25
Protocol Citation: Carolina Lopez 2024. SeV standard and copyback genomes PCR Protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvok3j7l4o/v1
Manuscript citation:
Pye SE, Achouri E, Yang Y, Musa A, López CB Validation of diverse and previously untraceable Sendai virus copyback viral genomes by direct RNA sequencing. Journal of Virology 99(8). doi: 10.1128/jvi.00894-25
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 28, 2024
Last Modified: January 05, 2025
Protocol Integer ID: 100788
Keywords: sendai virus, copyback genome, pcr protocols protocols for the detection, pcr protocols protocol, back genome, sev standard, pcr, sev, virus, copyback, genome
Abstract
Protocols for the detection of Sendai Virus (SeV) standard and copy-back genomes by PCR
Materials
Reverse Transcription (RT) **Different RT kits and steps for cbVG and Genome**
cbVG
Genome
PCR *Same kit and steps for both cbVG and Genome*
Troubleshooting
Before start
PRINCIPLES BEHIND THE PROCEDURE MUST BE UNDERSTOOD. PLEASE CONSULT WITH EXPERIENCED LAB MEMBER THE FIRST TIME YOU USE THIS PROCEDURE. UPDATE AS A GENERAL PROCEDURE AS NECESSARY BUT DO NOT MODIFY WITH SPECIFICS TO YOUR PROJECT, INSTEAD DOWNLOAD AND PASTE A MODIFIED COPY IN YOUR NOTEBOOK.
Reverse Transcription (RT)
10m
*Different RT kits and steps for cbVG and Genome
1/3) cbVG: Use SuperScript III Kit
Procedure
*RT reaction can be completed in a 10 µL reaction or a 20 µL reaction. The 10 µL reaction is preferred, if RNA concentrations allow, to help conserve reagents.
1. Start with 1 µg of RNA
10 µL reaction: dilute in 4 µL dH2O
20 µL reaction: dilute in 8 µL dH2O
2. Pre-mix 10 micromolar (µM) primer and dNTPs
10 µL reaction: 0.5 µL of each, per sample
20 µL reaction: 1 µL of each, per sample
3. Add Primer/dNTP mix to the RNA sample
10 µL reaction: 1 µL
20 µL reaction: 2 µL
4. Mix and spin down
5. Incubate at 65 °C for 00:10:00
Program in PCR Machine: RT-DI1
6. Prepare the mix (see TABLE below)
• Add the SIII enzyme separately to each sample, don’t include it in the mix.
7. Add mix to sample, vortex briefly, and spin down
10 µL reaction: 5 µL
20 µL reaction: 10 µL
8. Incubate at 50 °C for 00:50:00 then 85 °C for 00:05:00
Program in PCR Machine: RT-DI2
9. After program is finished keep at -20 °C or on ice for at least 00:05:00
————————————— Move to PCR Hood (for all later steps)
————————————— (Clean PCR hood with 10% bleach. Wipe down pipette with bleach in between samples.)
10. Add RNase H
10 µL reaction: 0.5 µL
20 µL reaction: 1 µL
11. Incubate at 37 °C for 00:20:00
Program in PCR Machine: RNaseH
12. Keep on ice for at least 00:05:00 then you can do the PCR or store at -20 °C
1h 35m
*Different RT kits and steps for cbVG and Genome
2/3) Genome: Use Roche Kit
Procedure
*RT reaction can be completed in a 10 µL reaction or a 20 µL reaction. The 10 ∝L reaction is preferred, if RNA concentrations allow, to help conserve reagents.
1. Start with 1000 ng of RNA diluted in dH2O
10 µL reaction: dilute in 3 µL dH2O
20 µL reaction: dilute in 6 µL dH2O
2. Add primer:
10 µL reaction: 3.5 µL
20 µL reaction: 7 µL
3. Incubate at 65 °C for 00:10:00
Program in PCR Machine: RT-DI1
4. Prepare the mix
5. Add mix to sample
10 µL reaction: 3.5 µL
20 µL reaction: 7 µL
6. Incubate at 50 °C for 60 minutes then 85 °C for 00:05:00 then 4 °C forever
Program in PCR machine: RORT2SPE
7. Keep in -20 °C for at least 00:20:00 before moving on to the PCR
35m
PCR
*Same kit and steps for both cbVG and Genome*
3/3) PCR
Procedure
1. Prepare master mix (can be done on bench) * Volumes below are for when 1 µL of cDNA is used. Adjust dH2O volume accordingly if more cDNA is added. Up to 4 µL cDNA can be added but increases background. Total reaction volume should equal 25 µL .
2. Add 24 µL of master mix to PCR tubes (can be done on bench)
3. Thaw cDNA samples and spin down in mini microcentrifuge
4. Add 1 µL of cDNA sample to master mix (add in PCR hood! cbVG are very stable!)
5. Run PCR
Program in PCR Machine: DIPCR
Gel Electrophoresis
1. Prepare 1% agarose gel
1 g pure agarose in 100 mL of 1X TAE buffer
Microwave until agarose solution dissolves completely (~3 min)
Let agarose solution cool before adding Ethidium bromide (if you can keep your fingers on the
flask without burning, then it is at an appropriate temperature)
Add 1-5 µL of Ethidium bromide and mix by swirling flask
2. Pour agarose solution into gel cast (remember to put in the well comb)
3. Let the agarose solidify (wait at least 00:30:00 )
4. Place gel in an electrophoresis chamber containing 1X TAE buffer (make sure buffer covers gel)
5. Load ladder and samples to wells
6 µL of Ladder
Ladder stock recipe:
100 µL Gene Ruler 100 bp Plus DNA ladder from Thermo Scientific: SM0321
100 µL DNA Gel Loading Dye (6X) from Thermo Scientific: R0611
400 µL dH2O
30 µL of Sample + Loading dye mix (dilutes from 6X to 1X)
5 µL of DNA Gel Loading Dye (6X) from Thermo Scientific: R0611
25 µL of sample
6. Run at 120 volts for 30-40 minutes
7. Analyze gel bands
30m
Sendai Virus Genomic and cbVG PCR CONCEPT
SeV copy back VG PCR strategy and validation. (A) Diagram of the genomic composition of the full-lengthSeV genome (gSeV) and of a representative copy-back VG of unknown length. Arrows indicate the location ofprimers used for RT and amplification (PCR). Full-length size of the genome is indicated. Expected amplicon size of 760 nt of the gSeV to be detected through our PCR assay is indicated. This strategy allows detection of most copy-back VGs replicating in an infected cell. (B) Schematics of SeV strain Cantell's predominant 546 nt long cbVG. Expected amplicon size of 278 nt of this particular cbVG to be detected through our PCR assay isindicated. (C) Validation of the cbVG PCR assay. cbVG and gSeV amplicons from plasmids encoding the full-length SeV strain Cantell genome (lane 1) or the SeV strain Cantell dominant cbVG (lane 2) after amplificationusing the primers depicted in (A).https://doi.org/10.1371/journal.ppat.1003703.
Note
The primers used to detect the Sendai virus genome, are designed between intergenomic regions, that are not transcribed which ensures that the genome pcr product is totally from genomic RNA and not mRNA.
The cbVG product shown is not the only cbVG that is presented in your samples, it’s just the one that was detected using these primers and this protocol sensitivity.
Same primers that are used in this protocol are the ones currently applied for Sev52 and SevC, SevCB as well.
Using random primers is not preferable as nonspecific products might be detected.
Protocol references
Hanaa Saleh 20230414