SARS‑CoV‑2 RNA Extraction using Biocomma Kit: Introduction, Reagent Preparation, Extraction Requirements, Procedure, and Keynotes

Introduction of SARS‑CoV‑2 RNA Extraction using Biocomma Kit

Biocomma nucleic acid (DNA/RNA) extraction kit is a Chinese product that is useful for RNA extraction systems for COVID-19 detection. It is applied for manual extraction and it is completed in four steps-

1. Lysis
2. Binding
3. Washing and
4. Elution.

A kit (for 50 tests) contains-

• Lysis buffer(GLX)-30 ml
• Proteinase K-20 mg/ml (1 ml)
• Spin column and collection tube(50/50 pieces)
• Wash buffer-1(PD)- 15 ml+15 ml isopropanol (outside)
• Wash buffer-2 ( PW)-15 ml+60 ml absolute ethanol(outside)
• RNase-free double distilled water – 10 ml (In stead of elution buffer)

Reagent preparation for SARS‑CoV‑2 RNA Extraction

1. Lysis buffer (GLX) is ready for use.

2. Add 15 ml Isopropanol to wash buffer-1(PD).

3. Similarly, add 60 ml isopropanol to wash buffer-2 ( PW).

4. Bring proteinase k from storage at -20C.

5. RNase-free ddH20-10 ml is for the elution step in place of the elution buffer.

Extraction Requirements

• Extraction kit
• Molecular Biology Grade Alcohol(Ethanol)
• Biological Safety Cabinet(BSC)-2
• Centrifuge
• Micropipettes and tips
• Waste containers
• Eppendorf tubes( 1.5 ml and 2.0 ml)
• Tissue paper rolls
• PPE sets
• Test specimen/nasopharyngeal swab/oropharyngeal swab

Extraction Procedure

1. Add 500 μL lysis buffer and 20 μL proteinase K in a centrifuge tube or Eppendorf tube for each test.

2. Now add 200μL sample.

4. Mix using a vortex for 1 minute and then incubate for 5-10 minutes.

6. Transfer the spin column (filter tube).

7. Centrifuge at 12000 rpm for 1 minute.

8. Discard filtrate.

9. Add 500 μL wash buffer-1 (PD).

10. Again centrifuge at 12000 rpm for 1 minute.

11. Discard filtrate.

12. Now put 700 μL wash buffer-2 (PW).

13. Centrifuge at 12000 rpm for 1 minute.

14. Discard filtrate.

15. Repeat steps 12,13 and 14.

16. Dry the spin column at 12000 rpm for 2 minutes.

17. Discard the tube (collection tube) from the spin column.

18. Keep the spin column/filter in a new centrifuge/ Eppendorf tube of 1.5 ml.

19. Add (RNase- free ddH20) 50- 100 μL slowly drop by drop at the Centre of the spin column so that it spreads easily over the column.

20. Finally centrifuge at 12000 rpm for 2 minutes.

21. Now throw the spin column and take the Eppendorf tube that has a template.

22. If delay in the master mix, maintain the cold chain.

Keynotes on SARS‑CoV‑2 RNA Extraction using Biocomma Kit

• This kit is useful for virus DNA/RNA Extraction (Spin Column).
• It can extract nucleic acid (DNA/RNA) from blood, tissue, organs, environmental sample, saliva, and nasal sample, and maximum remove impurities protein and other organic compound impurities.
• The extracted viral nucleic acid (DNA/RNA) can be directly used for downstream applications like PCR, RT-PCR, qPCR, and qRT-PCR experiment.
• The extraction procedure is simple, quick, and non-toxic with high-purity DNA/RNA and stable quality.

Bibliography

• https://www.biocomma.com/solutions/87.html
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7122149/