SARS-CoV-2 RNA Extraction: Introduction, Principle, Requirements and Extraction Procedure

SARS-CoV-2 RNA extraction

Introduction of SARS-CoV-2 

SARS-CoV-2 ( Severe acute respiratory syndrome coronavirus)  is a causative agent of  Coronavirus disease 2019 or COVID -19 is a viral disease whereas the provisional name 2019 novel coronavirus (2019-nCoV). It is a positive-sense single-stranded RNA virus and strain was first discovered in Wuhan, China, so it is sometimes referred to as the “Wuhan virus” or “Wuhan coronavirus”. Classification of this virus is as-

Realm:  Riboviria

Kingdom: Orthornavirae

Phylum: Pisuviricota

Order: Nidovirales

Family: Coronaviridae

Genus: Betacoronavirus

Subgenus: Sarbecovirus

Species: Severe acute respiratory syndrome-related coronavirus

Strain: Severe acute respiratory syndrome coronavirus 2

Primary reservoir bat whereas an intermediate animal reservoir such as a pangolin is also thought to be involved in its introduction to humans. The virus is primarily spread between people through close contact and via respiratory droplets produced from coughs or sneezes. It mainly enters human cells by binding to the receptor angiotensin-converting enzyme 2 (ACE2).

Principle of SARS-CoV-2 RNA extraction

RNA  is a polymeric substance present in SARS-CoV-2, consisting of a long single-stranded chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine, and uracil, which are bonded to the ribose sugar. RNA is used in all the steps of protein synthesis as well as carries the genetic information in this virus.

The isolation of RNA with high quality is a crucial step required to perform various molecular experiments. It is based on silica gel purification. The sample is lysed and digested with lysate and protease, RNA is released into the lysate. Transfer to an adsorption plate and filter column. RNA is adsorbed on the membrane, while protein is not adsorbed and is removed with filtration.

After washing proteins and other impurities are washed and RNA is finally eluted with low-salt buffer ( 10 MmTris, pH 8.0).

 

Requirements for SARS-CoV-2 RNA extraction

  • Kit contains-

Hipure viral mini-column ( Adsorption column)

PK/Carrier RNA (Proteinase/Poly A)

Protease Dissolve buffer( Glycerol/Tris/CaCl2)

Buffer VLF (Guanidine salt/ isopropanol)

Buffer CW (Tris/NaCl/Isopropanol)

Buffer AVE (Tris, pH 8.0)

  • Extra we need-

Biosafety cabinet 2A extraction room ( due to being an organism, SARS-CoV-2  of risk group 3)

  • Acceptable sample should have the following features-

Specimen Collection Methods

Collect specimen in virus transport medium (VTM).
Arrange for transportation in a way the sample is received within 24 hours.
Store collected specimen at 2 – 4° C (refrigerator) till ready for transportation.
NEVER STORE THE SAMPLE AT FREEZER COMPARTMENT(-20° C) as it may degrade the sample quality.

Transportation  Of Specimen

Transport specimen in 3 layers packaging system maintaining cold chain (2-4° C). Submit specimen with appropriate form fill up issued by WHO inappropriate testing site.

Sample Packaging for Coronavirus disease (COVID -19)

Layer 1: Put VTM with the specimen in a zip lock bag.
Layer 2: Place the zip lock bag in an airtight container lined with absorbent material(cotton).
Layer-3: Place this air-tight container in an icebox/vaccine box with an ice pack in all four corners as shown above image.
Sample rejection criteria if any 
The sample is transported without maintaining a cold chain.
The sample is received without proper form fill up.
Unlabeled sample.

  • Micropipettes and filter tips of following volumes-

10  µl

1000 µl (100-1000 µl adjustable)

40  µl

  • Microcentrifuge
  • Vortex mixer
  • Waste disposing of containers
  • Spray bottle with 70 % alcohol
  • The extractor should be with full personal protective equipment (PPE)
  • Tissues papers
  • Tube racks

Procedure of SARS-CoV-2 RNA extraction

  1. In a 1.5 ml centrifuge tube, add 400 µl buffer VLF and 10 µl PK/ carrier RNA.
  2. Transfer  200µl of sample to a centrifuge tube containing PK/ carrier RNA and mix by vortexing for 15 seconds. Leave at room temperature for 5 minutes.
  3.  Take out the new hipure viral mini-column and transfer the sample to the column. Centrifuge at 10, 000× g for 30-60 seconds.
  4. Discard the filtrate and place the column back into the collection tube. Add 500µl buffer CW to the column. Centrifuge at 10, 000× g for 30-60 seconds.
  5. Discard the filtrate and place the column back into the collection tube. Add 500µl buffer CW to the column. Centrifuge at 10, 000× g for 30-60 seconds.
  6. Discard the filtrate and place the column back into the collection tube. Centrifuge at 13, 000× g for 3 minutes to dry the column.
  7. Transfer the column to a new 1.5 ml centrifuge tube.
  8. Add 30-50 µl buffer AVE to the center of the membrane of the column. Centrifuge the column at 13, 000× g for 1 minute.
  9. Discard the column and use the RNA. If there is a delay of use, store the RNA at -80°C.

Further Readings

  1. Giaimo C (1 April 2020). “The Spiky Blob Seen Around the World”. The New York Times. Retrieved 6 April 2020.
  2. Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva AA, et al. (March 2020). “The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2”. Nature Microbiology. 5 (4): 536–544. doi:10.1038/s41564-020-0695-z. PMID 32123347. Archived from the original on 5 March 2020. Retrieved 3 March 2020.
  3. “Coronavirus disease named Covid-19”. BBC News Online. 11 February 2020. Archived from the original on 15 February 2020. Retrieved 15 February 2020.
  4. Surveillance case definitions for human infection with the novel coronavirus (nCoV): interim guidance v1, January 2020 (Report).World Health Organization. January 2020. hdl:10665/330376. WHO/2019-nCoV/Surveillance/v2020.1.
  5. “Q&A on coronaviruses (COVID-19)”. World Health Organization (WHO). 11 February 2020. Archived from the original on 20 January 2020. Retrieved 24 February 2020.
  6. “How COVID-19 Spreads”. U.S. Centers for Disease Control and Prevention (CDC). 27 January 2020. Archived from the original on 28 January 2020. Retrieved 29 January 2020.
  7. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200401-sitrep-72-covid-19.pdf?sfvrsn=3dd8971b_2
  8. https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html
  9. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance
  10. file:///C:/Users/Berry/Downloads/WHO-COVID-19-laboratory-2020.5-eng.pdf
  11. Brochure of manufacturer: Guangzhou Wondfo Biotech Co., Ltd

 

 

 

 

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