Mar 20, 2020
Generation of SARS-COV-2 RNA transcript standards for qRT-PCR detection assays
- Chantal Vogels1,
- Joseph Fauver1,
- Isabel M Ott1,
- Nathan D Grubaugh1
- 1Department of Epidemiology of Microbial Diseases, Yale School of Public Health
- Coronavirus Method Development Community
External link: https://doi.org/10.1371/journal.pone.0257474
Protocol Citation: Chantal Vogels, Joseph Fauver, Isabel M Ott, Nathan D Grubaugh 2020. Generation of SARS-COV-2 RNA transcript standards for qRT-PCR detection assays. protocols.io https://dx.doi.org/10.17504/protocols.io.bdv6i69e
Manuscript citation:
Vesga O, Agudelo M, Valencia-Jaramillo AF, Mira-Montoya A, Ossa-Ospina F, Ocampo E, Čiuoderis K, Pérez L, Cardona A, Aguilar Y, Agudelo Y, Hernández-Ortiz JP, Osorio JE (2021) Highly sensitive scent-detection of COVID-19 patients in vivo by trained dogs. PLoS ONE 16(9): e0257474. doi: 10.1371/journal.pone.0257474
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
Has been used to generate PCR-validated transcripts in our group.
Created: March 18, 2020
Last Modified: February 10, 2023
Protocol Integer ID: 34462
Keywords: SARS-CoV-2, COVID-19, qRT-PCR
Abstract
The protocol describes how to generate high-quality single-stranded RNA transcript standards (starting from a virus RNA stock) targeting the nsp10, RdRp, nsp14, envelope (E), and nucleocapsid (N) coding regions for use with China CDC, Hong Kong University (HKU), Corman et al. (Berlin), and US CDC SARS-CoV-2 primer and probe sets for qRT-PCR. (Sequences for transcripts generated, along with their corresponding assays, are provided under 'Guidelines.')
Guidelines
RNA transcript sequences
| Transcript name | Length (nt) | Genome position | Assays | Sequence | |
| SARS-COV-2 nsp10 RNA | 704 | 13,122 - 13,825 | China CDC-ORF1 | GUGGGGGACAACCAAUCACUAAUUGUGUUAAGAUGUUGUGUACACACACUGGUACUGGUCAGGCAAUAACAGUUACACCGGAAGCCAAUAUGGAUCAAGAAUCCUUUGGUGGUGCAUCGUGUUGUCUGUACUGCCGUUGCCACAUAGAUCAUCCAAAUCCUAAAGGAUUUUGUGACUUAAAAGGUAAGUAUGUACAAAUACCUACAACUUGUGCUAAUGACCCUGUGGGUUUUACACUUAAAAACACAGUCUGUACCGUCUGCGGUAUGUGGAAAGGUUAUGGCUGUAGUUGUGAUCAACUCCGCGAACCCAUGCUUCAGUCAGCUGAUGCACAAUCGUUUUUAAACGGGUUUGCGGUGUAAGUGCAGCCCGUCUUACACCGUGCGGCACAGGCACUAGUACUGAUGUCGUAUACAGGGCUUUUGACAUCUACAAUGAUAAAGUAGCUGGUUUUGCUAAAUUCCUAAAAACUAAUUGUUGUCGCUUCCAAGAAAAGGACGAAGAUGACAAUUUAAUUGAUUCUUACUUUGUAGUUAAGAGACACACUUUCUCUAACUACCAACAUGAAGAAACAAUUUAUAAUUUACUUAAGGAUUGUCCAGCUGUUGCUAAACAUGACUUCUUUAAGUUUAGAAUAGACGGUGACAUGGUACCACAUAUAUCACGUCAACGUCUUACUAAAUACACAAUGGCAGACCUCGUCU | |
| SARS-COV-2 RdRp RNA | 883 | 15,094 - 15,976 | Berlin-RdRp | AAUAGAGCUCGCACCGUAGCUGGUGUCUCUAUCUGUAGUACUAUGACCAAUAGACAGUUUCAUCAAAAAUUAUUGAAAUCAAUAGCCGCCACUAGAGGAGCUACUGUAGUAAUUGGAACAAGCAAAUUCUAUGGUGGUUGGCACAACAUGUUAAAAACUGUUUAUAGUGAUGUAGAAAACCCUCACCUUAUGGGUUGGGAUUAUCCUAAAUGUGAUAGAGCCAUGCCUAACAUGCUUAGAAUUAUGGCCUCACUUGUUCUUGCUCGCAAACAUACAACGUGUUGUAGCUUGUCACACCGUUUCUAUAGAUUAGCUAAUGAGUGUGCUCAAGUAUUGAGUGAAAUGGUCAUGUGUGGCGGUUCACUAUAUGUUAAACCAGGUGGAACCUCAUCAGGAGAUGCCACAACUGCUUAUGCUAAUAGUGUUUUUAACAUUUGUCAAGCUGUCACGGCCAAUGUUAAUGCACUUUUAUCUACUGAUGGUAACAAAAUUGCCGAUAAGUAUGUCCGCAAUUUACAACACAGACUUUAUGAGUGUCUCUAUAGAAAUAGAGAUGUUGACACAGACUUUGUGAAUGAGUUUUACGCAUAUUUGCGUAAACAUUUCUCAAUGAUGAUACUCUCUGACGAUGCUGUUGUGUGUUUCAAUAGCACUUAUGCAUCUCAAGGUCUAGUGGCUAGCAUAAAGAACUUUAAGUCAGUUCUUUAUUAUCAAAACAAUGUUUUUAUGUCUGAAGCAAAAUGUUGGACUGAGACUGACCUUACUAAAGGACCUCAUGAAUUUUGCUCUCAACAUACAAUGCUAGUUAAACAGGGUGAUGAUUAUGUGUACCUUCCUUACCCAGAUCCAUCAAGAAUCCUAGGGGCCGGCUGUUUUGUAGAUG | |
| SARS-COV-2 nsp14 RNA | 848 | 18,447 - 19,294 | HKU-ORF1 | UAGUGCUAAACCACCGCCUGGAGAUCAAUUUAAACACCUCAUACCACUUAUGUACAAAGGACUUCCUUGGAAUGUAGUGCGUAUAAAGAUUGUACAAAUGUUAAGUGACACACUUAAAAAUCUCUCUGACAGAGUCGUAUUUGUCUUAUGGGCACAUGGCUUUGAGUUGACAUCUAUGAAGUAUUUUGUGAAAAUAGGACCUGAGCGCACCUGUUGUCUAUGUGAUAGACGUGCCACAUGCUUUUCCACUGCUUCAGACACUUAUGCCUGUUGGCAUCAUUCUAUUGGAUUUGAUUACGUCUAUAAUCCGUUUAUGAUUGAUGUUCAACAAUGGGGUUUUACAGGUAACCUACAAAGCAACCAUGAUCUGUAUUGUCAAGUCCAUGGUAAUGCACAUGUAGCUAGUUGUGAUGCAAUCAUGACUAGGUGUCUAGCUGUCCACGAGUGCUUUGUUAAGCGUGUUGACUGGACUAUUGAAUAUCCUAUAAUUGGUGAUGAACUGAAGAUUAAUGCGGCUUGUAGAAAGGUUCAACACAUGGUUGUUAAAGCUGCAUUAUUAGCAGACAAAUUCCCAGUUCUUCACGACAUUGGUAACCCUAAAGCUAUUAAGUGUGUACCUCAAGCUGAUGUAGAAUGGAAGUUCUAUGAUGCACAGCCUUGUAGUGACAAAGCUUAUAAAAUAGAAGAAUUAUUCUAUUCUUAUGCCACACAUUCUGACAAAUUCACAGAUGGUGUAUGCCUAUUUUGGAAUUGCAAUGUCGAUAGAUAUCCUGCUAAUUCCAUUGUUUGUAGAUUUGACACUAGAGUGCUAUCUAACCUUAACUUGCCUGGUUGUGAUGGUGGCAGUU | |
| SARS-COV-2 envelope (E) RNA | 808 | 26,207 - 27,116 | Berlin-E | GCGUGCCUUUGUAAGCACAAGCUGAUGAGUACGAACUUAUGUACUCAUUCGUUUCGGAAGAGACAGGUACGUUAAUAGUUAAUAGCGUACUUCUUUUUCUUGCUUUCGUGGUAUUCUUGCUAGUUACACUAGCCAUCCUUACUGCGCUUCGAUUGUGUGCGUACUGCUGCAAUAUUGUUAACGUGAGUCUUGUAAAACCUUCUUUUUACGUUUACUCUCGUGUUAAAAAUCUGAAUUCUUCUAGAGUUCCUGAUCUUCUGGUCUAAACGAACUAAAUAUUAUAUUAGUUUUUCUGUUUGGAACUUUAAUUUUAGCCAUGGCAGAUUCCAACGGUACUAUUACCGUUGAAGAGCUUAAAAAGCUCCUUGAACAAUGGAACCUAGUAAUAGGUUUCCUAUUCCUUACAUGGAUUUGUCUUCUACAAUUUGCCUAUGCCAACAGGAAUAGGUUUUUGUAUAUAAUUAAGUUAAUUUUCCUCUGGCUGUUAUGGCCAGUAACUUUAGCUUGUUUUGUGCUUGCUGCUGUUUACAGAAUAAAUUGGAUCACCGGUGGAAUUGCUAUCGCAAUGGCUUGUCUUGUAGGCUUGAUGUGGCUCAGCUACUUCAUUGCUUCUUUCAGACUGUUUGCGCGUACGCGUUCCAUGUGGUCAUUCAAUCCAGAAACUAACAUUCUUCUCAACGUGCCACUCCAUGGCACUAUUCUGACCAGACCGCUUCUAGAAAGUGAACUCGUAAUCGGAGCUGUGAUCCUUCGUGGACAUCUUCGUAUUGCUGGACACCAUCUAGGACGCUGUGACAUCAAGGACCUGCC | |
| SARS-COV-2 nucleocapsid (N) RNA | 1363 | 28,068 - 29,430 | China CDC-N; HKU-N; US CDC-N1; US CDC-N2; US CDC-N3 | GAAUUGUGCGUGGAUGAGGCUGGUUCUAAAUCACCCAUUCAGUACAUCGAUAUCGGUAAUUAUACAGUUUCCUGUUUACCUUUUACAAUUAAUUGCCAGGAACCUAAAUUGGGUAGUCUUGUAGUGCGUUGUUCGUUCUAUGAAGACUUUUUAGAGUAUCAUGACGUUCGUGUUGUUUUAGAUUUCAUCUAAACGAACAAACUAAAAUGUCUGAUAAUGGACCCCAAAAUCAGCGAAAUGCACCCCGCAUUACGUUUGGUGGACCCUCAGAUUCAACUGGCAGUAACCAGAAUGGAGAACGCAGUGGGGCGCGAUCAAAACAACGUCGGCCCCAAGGUUUACCCAAUAAUACUGCGUCUUGGUUCACCGCUCUCACUCAACAUGGCAAGGAAGACCUUAAAUUCCCUCGAGGACAAGGCGUUCCAAUUAACACCAAUAGCAGUCCAGAUGACCAAAUUGGCUACUACCGAAGAGCUACCAGACGAAUUCGUGGUGGUGACGGUAAAAUGAAAGAUCUCAGUCCAAGAUGGUAUUUCUACUACCUAGGAACUGGGCCAGAAGCUGGACUUCCCUAUGGUGCUAACAAAGACGGCAUCAUAUGGGUUGCAACUGAGGGAGCCUUGAAUACACCAAAAGAUCACAUUGGCACCCGCAAUCCUGCUAACAAUGCUGCAAUCGUGCUACAACUUCCUCAAGGAACAACAUUGCCAAAAGGCUUCUACGCAGAAGGGAGCAGAGGCGGCAGUCAAGCCUCUUCUCGUUCCUCAUCACGUAGUCGCAACAGUUCAAGAAAUUCAACUCCAGGCAGCAGUAGGGGAACUUCUCCUGCUAGAAUGGCUGGCAAUGGCGGUGAUGCUGCUCUUGCUUUGCUGCUGCUUGACAGAUUGAACCAGCUUGAGAGCAAAAUGUCUGGUAAAGGCCAACAACAACAAGGCCAAACUGUCACUAAGAAAUCUGCUGCUGAGGCUUCUAAGAAGCCUCGGCAAAAACGUACUGCCACUAAAGCAUACAAUGUAACACAAGCUUUCGGCAGACGUGGUCCAGAACAAACCCAAGGAAAUUUUGGGGACCAGGAACUAAUCAGACAAGGAACUGAUUACAAACAUUGGCCGCAAAUUGCACAAUUUGCCCCCAGCGCUUCAGCGUUCUUCGGAAUGUCGCGCAUUGGCAUGGAAGUCACACCUUCGGGAACGUGGUUGACCUACACAGGUGCCAUCAAAUUGGAUGACAAAGAUCCAAAUUUCAAAGAUCAAGUCAUUUUGCUGAAUAAGCAUAUUGACGCAUACAAAACAUUCCCACCAACAGAGCCUAAAAAGGACAAAAAGAAGAAGGCUGAUGAAACUCAAGCCUUACCGCAGAGACA |
RNA transcripts generated by this protocol, along with their length (in nucleotides), their position on the SARS-COV-2 reference genome, and the assays they serve as positive controls for (since all assays are multiplexed, the target is specified).
Materials
MATERIALS
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
Agilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
Bioanalyzer RNA 6000 Nano KitAgilent TechnologiesCatalog #5067
SuperScript™ IV VILO™ Master MixThermo FisherCatalog #11756500
MEGAscript™ T7 Transcription KitThermo FisherCatalog #AM1334
Qubit™ dsDNA HS Assay KitThermo FisherCatalog #Q32851
Qubit™ RNA HS Assay KitThermo FisherCatalog #Q32852
Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01
STEP MATERIALS
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01
Qubit™ dsDNA HS Assay KitThermo FisherCatalog #Q32851
Agilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
MEGAscript™ T7 Transcription KitThermo FisherCatalog #AM1334
Qubit™ RNA HS Assay KitThermo FisherCatalog #Q32852
Bioanalyzer RNA 6000 Nano KitAgilent TechnologiesCatalog #5067
SuperScript™ IV VILO™ Master MixThermo FisherCatalog #11756500
Protocol materials
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
MEGAscript™ T7 Transcription KitThermo FisherCatalog #AM1334
Qubit™ dsDNA HS Assay KitThermo FisherCatalog #Q32851
Qubit™ RNA HS Assay KitThermo FisherCatalog #Q32852
Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
Agilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
Qubit™ RNA HS Assay KitThermo FisherCatalog #Q32852
Bioanalyzer RNA 6000 Nano KitAgilent TechnologiesCatalog #5067
Bioanalyzer RNA 6000 Nano KitAgilent TechnologiesCatalog #5067
Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01
SuperScript™ IV VILO™ Master MixThermo FisherCatalog #11756500
Agilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
Qubit™ dsDNA HS Assay KitThermo FisherCatalog #Q32851
MEGAscript™ T7 Transcription KitThermo FisherCatalog #AM1334
SuperScript™ IV VILO™ Master MixThermo FisherCatalog #11756500
SuperScript™ IV VILO™ Master MixThermo FisherCatalog #11756500
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01
Qubit™ dsDNA HS Assay KitThermo FisherCatalog #Q32851
Agilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
MEGAscript™ T7 Transcription KitThermo FisherCatalog #AM1334
Qubit™ RNA HS Assay KitThermo FisherCatalog #Q32852
Bioanalyzer RNA 6000 Nano KitAgilent TechnologiesCatalog #5067
Safety warnings
Please be aware that full length single-stranded positive sense SARS-CoV-2 RNA is considered infectious and must be handled in BSL-2 or above conditions. Please consult with your institutional biosafety team before handling SARS-CoV-2 RNA.
Before start
Basic outline of steps: cDNA synthesis > PCR > Purification > Quality + Quantity check > T7 RNA transcription > Purification > Quality + Quantity check
Preparation of cDNA
Preparation of cDNA
Isolate viral RNA using Omega Viral DNA/RNA kit, Trizol, or equivalent.
Many different cDNA synthesis kits can be used, but choose something that is relatively high-fidelity. The current protocol uses SuperScript IV VILO Master Mix because the enzyme has low error rates and the protocol is fast and easy.
SuperScript™ IV VILO™ Master MixThermo FisherCatalog #11756500
| Component | Volume in 20 µL reaction | |
| SSIV VILO Master Mix | 4 µL | |
| Nuclease-free water | 11 µL | |
| Virus RNA | 5 µL |
Run the following cycles on a thermocycler:
| Temperature | Time | |
| 25°C | 10 minutes | |
| 50°C | 10 minutes | |
| 85°C | 5 minutes | |
| 4°C | ∞ |
Store samples at 4 °C (same day) or -20 °C (up to a week) until ready for PCR.
PCR
PCR
Prepare the following primers at a concentration of 10 µM, depending on which assay is needed. The nucleocapsid (N) transcript is for the US CDC-N1, N2, and N3 (N3 is not currently in use), HKU-N, and China CDC-N assays. The envelope (E) transcript is for use with the Berlin-E assay, nsp14 transcript is for use with the HKU-ORF1 assay, the RdRp transcript is for use with the Berlin RdRp assay (low sensitivity), and the nsp10 transcript is for use with the China CDC-ORF1 assay. See here for assay primer sequences and references.
| Target | Primer | Sequence | Position | Amplicon size | |
| N | N-Std-T7-Fwd | TAATACGACTCACTATAGGGGAATTGTGCGTGGATGAGGC | 28068 | 1363 | |
| N-Std-Rev | TGTCTCTGCGGTAAGGCTTG | 29411 | |||
| E | E-Std-T7-Fwd | TAATACGACTCACTATAGGGGCGTGCCTTTGTAAGCACAA | 26207 | 808 | |
| E-Std-Rev | GGCAGGTCCTTGATGTCACA | 27016 | |||
| nsp14 | nsp14-Std-T7-Fwd | TAATACGACTCACTATAGGGTAGTGCTAAACCACCGCCTG | 18447 | 848 | |
| nsp14-Std-Rev | AACTGCCACCATCACAACCA | 19275 | |||
| RdRp | RdRp-Std-T7-Fwd | TAATACGACTCACTATAGGGAATAGAGCTCGCACCGTAGC | 15094 | 883 | |
| RdRp-Std-Rev | CATCTACAAAACAGCCGGCC | 15957 | |||
| nsp10 | nsp10-Std-T7-Fwd | TAATACGACTCACTATAGGGGTGGGGGACAACCAATCACT | 13122 | 704 | |
| nsp10-Std-Rev | AGACGAGGTCTGCCATTGTG | 13806 |
Primers for PCR amplification. Note that forward primers include a T7 promoter sequence.
Prepare the following PCR reaction for each sample:
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
| Component | Volume in 25 µL reaction | |
| Q5 2x Master Mix | 12.5 µL | |
| 10 µM Primer F-T7 | 1.25 µL | |
| 10 µM Primer R | 1.25 µL | |
| Nuclease-free water | 8 µL | |
| cDNA | 2 µL |
Run the following cycles on a thermocycler:
| Temperature | Time | |
| 98°C | 30 seconds | |
| 98°C | 10 seconds | |
| 55°C | 30 seconds | |
| 72°C | 90 seconds | |
| Repeat steps 2-4 for a total of 35 cycles | ||
| 72°C | 2 minutes | |
| 4°C | ∞ | |
Post-PCR cleanup
Post-PCR cleanup
Allow Mag-Bind TotalPure NGS beads to equilibrate to room temperature, vortex until homogenous.
Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01
Bring PCR product volume up to 25 µL with water (if not at volume already).
Add 20 µL of beads to 25 µL of PCR product, mix well, and incubate at room temperature for 10 minutes.
Note
If your lab has a KingFisher extractor to use for post-PCR cleanup steps, our automated protocols are available here: https://github.com/grubaughlab/Kingfisher_protocols (use ‘purification.bdz’ for this cleanup)
Place tubes on a magnetic stand and incubate until solution appears clear.
Discard supernatant without disturbing the beads.
While tubes are on the magnet, add 200 µL of 80% ethanol, incubate for 30 seconds, and discard the ethanol wash.
Repeat previous 80% ethanol wash and remove as much ethanol as possible.
Leave tubes on magnet and air dry for 5 minutes.
Remove tubes from magnet and add 20 µL of nuclease-free water. Mix well by pipetting.
Place tubes on magnet stand. When solution appears clear, remove supernatant without disturbing the beads and place into new tubes.
Intermediate quantification
Intermediate quantification
Quantify the DNA concentration using the Qubit High Sensitivity DNA kit (or equivalent) from 1 µL of each product, following manufacturer's protocol.
Qubit™ dsDNA HS Assay KitThermo FisherCatalog #Q32851
Check DNA fragment distributions of the samples using the BioAnalyzer DNA 1000 kit, following manufacturer's protocol.
Agilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
Reverse Transcription (Megascript T7 kit)
Reverse Transcription (Megascript T7 kit)
Important: thaw reagents on ice, but keep the 10X reaction buffer at room temperature while assembling the reaction. Add the 10X reaction buffer after water and NTPs have been added to the tube.
MEGAscript™ T7 Transcription KitThermo FisherCatalog #AM1334
Mix equal volumes (2 µL/sample) of the four ribonucleotide solutions together and add 8 µL of the mixture for each sample.
Use 2 µL of pTRI-Xef in a standard reaction as a transcription control. This should yield a transcript of 1.89 kb.
| Component | Volume in 20 µL reaction | |
| Nuclease-free water | Add to 20 µL | |
| NTP mixture (ATP + CTP + GTP + UTP) | 8 µL | |
| 10X reaction buffer | 2 µL | |
| Purified PCR product | 0.1-0.2 µg PCR product | |
| Enzyme mix | 2 µL |
Mix by pipetting up and down and microfuge briefly to collect reaction mixture at the bottom of the tube.
Incubate at 37 °C for 4 hours .
Add 1 µL of TURBO DNase, mix well and incubate 37 °C for 15 minutes .
RNA product purification
RNA product purification
Add the following components to a 1.5 mL tube.
| Component | Volume in 81 µL reaction | |
| Nuclease-free water | 30 µL | |
| Lithium Chloride | 30 µL | |
| T7 transcription product | 21 µL |
Mix and chill at -20 °C for >30 minutes .
Centrifuge at 4 °C for 15 minutes at maximum speed to pellet the RNA.
Carefully remove supernatant. Wash pellet with 1 mL of 70% ethanol and re-centrifuge at 4 °C for 15 minutes at maximum speed to maximize removal of unincorporated nucleotides.
Carefully remove the ethanol and resuspend in 20 µL of nuclease-free water.
Final quantification
Final quantification
Quantify the RNA concentration using the Qubit High Sensitivity RNA kit from 1 µL of each product, following manufacturer's protocol.
Qubit™ RNA HS Assay KitThermo FisherCatalog #Q32852
Check RNA fragment distributions of the samples using the BioAnalyzer RNA 6000 pico kit, following manufacturer's protocol.
Bioanalyzer RNA 6000 Nano KitAgilent TechnologiesCatalog #5067
Use the following RNA copy number calculator to calculate genome equivalents per uL and prepare standards.