Isolation and Amplification of SARS-CoV-2 RNA from Nasopharyngeal Specimen
Frank Twum Aboagye, Maame Ekua Acquah
RNA
RNA Extraction
SARS-CoV-2
Zymo
COVID-19
RT-PCR
Virus
Nasopharyngeal specimen
Viral Transport Medium
Disclaimer
This is an optimised protocol for SARS-CoV-2 RNA isolation and amplification using Zymo Quick-RNA Viral Kit (200 prep) and Allplex 2019nCoV Assay Kit. The authors do not accept any liability for the collection and handling of both samples and reagents, results from the use of the protocol and its interpretation as well as any errors or omissions that may be made. The reader should make his/her own evaluation as to the appropriateness of the procedures described.
Abstract
COVID-19 caused by the SARS-CoV-2 was declared a global pandemic by the World Health Organization in March 2020. Classical symptoms associated with the infection include fever, cough, chills, headache, and muscle aches amongst others. To effectively diagnose the infection and contain its spread, efficient diagnostic tools are required. The current gold standard for the confirmation of SARS-CoV-2 infection is RT-PCR, this protocol outlines procedures for the isolation and amplification of SARS-CoV-2 RNA from Nasopharyngeal specimens using the Zymo Quick-RNA Viral Kit and Allplex 2019nCoV Assay Kit respectively.
Before start
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DTT is not stable in solution. Only freshly-made DTT solutions should be used
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Aliquot the needed volume of reagents from the stock for the procedure to prevent contamination of the large reagent stock.
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Allow the amplification kit to thaw completely in a 4°C fridge, before preparing the PCR master mix. Avoid centrifuging the reagents to defrost them.
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Decontaminate all workspace with 1% bleach followed by 70% alcohol
Steps
Reagent Preparation - Viral RNA Wash Buffer
To each bottle of 48mL of RNA Wash Buffer (concentrate) add 192mL of molecular grade ethanol (95-100%).
Invert the reagent bottle several times and label with the date of preparation
Reagent Preparation - Viral RNA Buffer
Weigh 0.75g of dithiothreitol (DTT) into 15mL of nuclease-free water
Allow the pellets to dissolve completely.
Add 12mL of the freshly prepared DTT solution to the 100 mL Viral RNA Buffer (concentrate). Invert several times to mix completely.
RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
Vortex the specimen for 0h 0m 30s at 3000rpm,0h 0m 0s and allow it to sit on the bench for 1 minute
Aliquot 200µL of the specimen into a sterile 1.5 mL microcentrifuge tube
Add to the specimen 400µL of the RNA Buffer and vortex at 3000rpm
Transfer the solution in step 3 into a Zymo-Spin™ IC Column in a clean collection tube and centrifuge at 8000rpm
Discard the flow-through liquid and transfer the Zymo-Spin™ IC Column into a new collection tube.
Transfer into the Zymo-Spin™ IC Column 500µL of RNA Wash Buffer (refer to reagent preparation for reconstitution of RNA Wash Buffer).
Centrifuge the Zymo-Spin™ IC Column in the collection tube at 10000rpm and discard the flow-through liquid. Repeat this step.
Add500µL of absolute ethanol to the Zymo-Spin™ IC Column and centrifuge at 10000rpm and discard the flow-through liquid.
Perform a dry-spin step at 13000rpm rpm to eliminate any trace of ethanol.
Transfer the Zymo-Spin™ IC Column into a new sterile 1.5 mL microcentrifuge tube and add to the spin column 25 μL of nuclease-free water.
Centrifuge the spin column for 1 minute at 8000 rpm. Cover the microcentrifuge tube while inspecting for the elute
Store the RNA at -20°C pending downstream analysis.
Preparation of PCR Master Mix (AllplexTM 2019nCoV Assay Kit)
| A | B | C |
|---|---|---|
| Reagent | X 1 (μL) | X 10 (μL) |
| 2019 nCoV MuDT* Ol igo M ix (MOM) | 5.0 | 50.0 |
| RNase Free Water (PCR Grade) | 5.0 | 50.0 |
| Real-t ime One-step Buffer | 5.0 | 50.0 |
| Real-t ime One-step Enzyme | 2.0 | 20.0 |
| Total Volume | 17.0 | 170.0 |
MuDT is the brand name of Seegene’s oligo mixtureNB:1. Add the One-step Enzyme as the last reagent and pipette up and down several times to wash the reagent out of the tip since it is slightly viscous.2. Pulse vortex the master mix and centrifuge at 3000 rpm for 30 seconds.
Preparation of RT-PCR Reaction Mix
| A | B | C |
|---|---|---|
| Reagent | X 1 (μL) | X 10 (μL) |
| 2019 nCoV MuDT* Ol igo M ix (MOM) | 5.0 | 50.0 |
| RNase Free Water (PCR Grade) | 5.0 | 50.0 |
| Real-t ime One-step Buffer | 5.0 | 50.0 |
| Real-t ime One-step Enzyme | 2.0 | 20.0 |
| Viral RNA | 8.0 | - |
| Total Reaction Volume | 25.0 |
MuDT is the brand name of Seegene’s ol igo mixtureNB: 1. Add 4 μL of the exogenous internal control (IC) to the extracted RNA immediately before pipetting the required volume for the reaction.2. Centrifuge the qPCR plate or 8-strip qPCR tube for 30 seconds at 1000 rpm
RT-PCR Amplification Protocol (Allplex 2019nCoV Assay Kit)
Cycling Conditions : Reverse transcription at 50°C for 20 minutes, followed by an initial denaturation at 95°C for 15 minutes and 45 cycles of 94°C for 15 seconds and 58°C for 30 seconds (plate read: data acquisition)
Fluorophores : FAM (E gene), Cal Red 610 (RdRP gene), Quasar 670 (N gene), and HEX (Internal Control).
Cut-off Ct-value : 40
