TITLE: Isolation and Amplification of SARS-CoV-2 RNA from Nasopharyngeal Specimen
AUTHORS: Frank Twum Aboagye, Maame Ekua Acquah

[DESCRIPTION]
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]
1. DTT is not stable in solution. Only freshly-made DTT solutions should be used

2. Aliquot the needed volume of reagents from the stock for the procedure to prevent contamination of the large reagent stock.

3. 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.

4. Decontaminate all workspace with 1% bleach followed by 70% alcohol

[GUIDELINES]
Viral RNA Wash Buffer Preparation
Viral RNA Buffer Preparation
RNA Isolation Procedure (Zymo Quick-RNA Viral Kit)
RT-PCR Mix Preparation Procedure (Allplex 2019nCoV Assay Kit)
RT-PCR Reaction Mix Preparation Procedure
RT-PCR Amplification Protocol (Allplex 2019nCoV Assay Kit)

[STEPS]
SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
6. Vortex the specimen for 30 s at 3000 rpm and allow it to sit on the bench for 1 minute

SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
7. Aliquot 200 µL of the specimen into a sterile 1.5 mL microcentrifuge tube

SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
8. Add to the specimen 400 µL of the RNA Buffer and vortex at 3000 rpm, 30 s
SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
9. Transfer the solution in step 3 into a Zymo-Spin™ IC Column in a clean collection tube and centrifuge at 8000 rpm, 2 min
SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
10. Discard the flow-through liquid and transfer the Zymo-Spin™ IC Column into a new collection tube.
SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
11. Transfer into the  Zymo-Spin™ IC Column 500 µL of RNA Wash Buffer (refer to reagent preparation for reconstitution of RNA Wash Buffer).

SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
12. Centrifuge the Zymo-Spin™ IC Column in the collection tube at 10000 rpm, 30 s and discard the flow-through liquid. Repeat this step.
SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
13. Add500 µL of absolute ethanol to the Zymo-Spin™ IC Column and centrifuge at 10000 rpm, 1 min and discard the flow-through liquid.

SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
14. Perform a dry-spin step at 13000 rpm, 1 min rpm to eliminate any trace of ethanol.

SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
15. 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.
SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
16. Centrifuge the spin column for 1 minute at 8000 rpm. Cover the microcentrifuge tube while inspecting for the elute
SECTION: RNA Extraction Protocol (Zymo Quick-RNA Viral Kit)
17. Store the RNA at -20 °C pending downstream analysis. 

SECTION: Reagent Preparation - Viral RNA Wash Buffer
1. To each bottle of 48 mL of RNA Wash Buffer (concentrate) add 192 mL of molecular grade ethanol (95-100%).

SECTION: Reagent Preparation - Viral RNA Buffer
3. Weigh 0.75 g of dithiothreitol (DTT) into 15 mL of nuclease-free water

SECTION: Reagent Preparation - Viral RNA Wash Buffer
2. Invert the reagent bottle several times and label with the date of preparation
SECTION: Reagent Preparation - Viral RNA Buffer
4. Allow the pellets to dissolve completely.
SECTION: Reagent Preparation - Viral RNA Buffer
5. Add 12 mL of the freshly prepared DTT solution to the 100 mL Viral RNA Buffer (concentrate). Invert several times to mix completely.
 


SECTION: Preparation of PCR Master Mix (AllplexTM 2019nCoV Assay Kit)
18. 
 

SECTION: Preparation of RT-PCR Reaction Mix
19. 


 

SECTION: RT-PCR Amplification Protocol (Allplex 2019nCoV Assay Kit)
20. 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