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Extraction, Purification and Detection of SARS-CoV-2 Nucleic Acid

SL Shanghai BioGerm Medical Technology Co. LTD

Published: Sep 5, 2020 DOI: 10.21769/BioProtoc.5000 Views: 2522

Reviewed by: Amr Galal Abdelraheem IbrahimPayton Tung-On YauMigla Miskinyte

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Abstract

Nucleic acid extraction and purification kit is used for isolating high-quality pathogen nucleic acids (DNA/RNA) from a variety of specimens. Nucleic acid can be directly extracted from liquid samples such as blood, serum, plasma, urine, nasopharyngeal swabs, and cell culture. And soil samples such as stool, tissue samples need to be pre- treated, obtaining a supernatant and carrying out extraction and purification to obtain the nucleic acids. The obtained nucleic acids can be directly used in related experiments such as Fluorescence quantitative PCR, RT-PCR, biochip analysis, second-generation sequencing.

The SARS-CoV-2 Nucleic Acid Detection Kit is a molecular in vitro diagnostic test that aids in the detection and diagnosis of 2019-nCoV and is based on widely used nucleic acid amplification technology. The product contains oligonucleotide primers and dual-labeled hydrolysis probes and control material used in RT-PCR for the in vitro qualitative detection of SARS-CoV-2 RNA in nasopharyngeal swab, oropharyngeal swab and sputum specimens.

The oligonucleotide primers and probes for detection of SARS-CoV-2 were selected from regions of the virus ORF1ab gene and nucleocapsid (N) gene. The panel is designed for specific detection of the SARS-CoV-2 (two primer/probe sets). An additional primer/probe set to detect the human RNase P gene (RNP) in control samples and clinical specimens is also included in the panel.

Background

Nucleic acid extraction and purification kit is based on superparamagnetic particles technology with high adhesion. Under the condition of high concentration of ionizing agent, the nanometer magnetic particles can specifically adsorb nucleic acids through hydrogen bonding and electrostatic, while proteins or other impurities are not absorbed. The nucleic acid-adsorbed nanoparticles are washed to remove proteins, salts and other impurities on the particles surface by washing buffer. Finally, the low-salt buffer can be used to elute the purified nucleic acids on the particles. The magnetic beads with special surface modification have stronger ability of binding nucleic acid and easier elution, which can minimize the risk of cross- contamination and improve detection sensitivity and accuracy. The kit has simple operation steps in a short time, and can complete the entire extraction process at room temperature (about 25 °C).

SARS-CoV-2 Nucleic Acid Detection Kit contains the assays and controls for a real-time reverse transcription polymerase chain reaction (RT-PCR) test intended for the qualitative detection of nucleic acid from SARS-CoV-2 in nasopharyngeal swab, oropharyngeal swab, sputum specimens from individuals suspected of COVID-19. Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests.

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in nasopharyngeal swab, oropharyngeal swab, sputum during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results are indicative of active infection. Laboratories within the United States and its territories are required to report all positive results to the appropriate public health authorities. Negative results do not preclude 2019-nCoV infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.

The SARS-CoV-2 Nucleic Acid Detection Kit is intended for use by qualified and trained clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures. SARS-CoV-2 Nucleic Acid Detection Kit is only for use under the Food and Drug Administration’s Emergency Use Authorization.

Materials and Reagents

Materials

  1. Dedicated laboratory coat for each area
  2. Disposable booties
  3. Biohazard bag for tip and tube disposal
  4. Powder-free latex, vinyl or nitrile gloves
  5. 1.5 ml screw-capped microcentrifuge tubes
  6. Aerosol barrier tips
  7. 96-well PCR plates
  8. Optical caps
  9. Disposable Plasticware
    The use of sterile, disposable polypropylene tubes is recommended throughout the procedure. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases
  10. 20% (v/v) bleach solution (2.0% w/v sodium hypochlorite in water)
  11. 70% ethanol
  12. Nuclease-free water
  13. Qiagen extractor EZ1 Advanced XL and EZ1 DSP Virus kit (62724)

Kits
  1. Nucleic acid extraction and purification kit
    Notes:
    1. Date of manufacture and expiry: see outer packing box.
    2. Transport and store at room temperature (about 25 °C), and the validity period is 6 months.

    1. Nucleic acid extraction and purification kit (25 reactions) (Shanghai BioGerm Medical Technology Co., LTD, catalog number: TQ-BG001-025)
      1. Beads* (Reagent number: BG001-PA1, 0.8 ml)
      2. Lysate (Reagent number: BG001-PA2, 15 ml)
      3. Washing buffer I** (Reagent number: BG001-PA3, 6.6 ml)
        Note: Need add 8.4 ml anhydrous ethanol before use.
      4. Washing buffer II** (Reagent number: BG001-PA4, 6 ml)
        Note: Need add 24 ml anhydrous ethanol before use.
    2. Nucleic acid extraction and purification kit (50 reactions) (Shanghai BioGerm Medical Technology Co., LTD, catalog number: TQ-BG001-050) 
      1. Beads* (Reagent number: BG001-PD1, 1.7 ml)
      2. Lysate (Reagent number: BG001-PD2, 30 ml)
      3. Washing buffer I** (Reagent number: BG00 1-PD3, 17 ml)
        Note: Need add 22 ml anhydrous ethanol before use.
      4. Washing buffer II** (Reagent number: BG00 1-PD4, 15 ml)
        Note: Need add 60 ml anhydrous ethanol before use.
      5. Eluate buffer (Reagent number: BG001-PD5, 25 ml)
    3. Nucleic acid extraction and purification kit (100 reactions) (Shanghai BioGerm Medical Technology Co., LTD, catalog number: TQ-BG001-100)
      1. Beads* (Reagent number: BG001-PB1, 3.3 ml)
      2. Lysate (Reagent number: BG001-PB2, 50 ml)
      3. Washing buffer I** (Reagent number: BG001-PB3, 33 ml)
        Note: Need add 42 ml anhydrous ethanol before use.
      4. Washing buffer II** (Reagent number: BG00 1-PB4, 25 ml)
        Note: Need add 100 ml anhydrous ethanol before use.
      5. Eluate buffer (Reagent number: BG001-PB5, 50 ml)
    4. Nucleic acid extraction and purification kit (200 reactions) (Shanghai BioGerm Medical Technology Co., LTD, catalog number: TQ-BG001-200)
      1. Beads* (Reagent number: BG001-PC1, 6.6 ml)
      2. Lysate (Reagent number: BG001-PC2, 100 ml)
      3. Washing buffer I** (Reagent number: BG001-PC3, 66 ml)
        Note: Need add 84 ml anhydrous ethanol before use.
      4. Washing buffer II** (Reagent number: BG00 1-PC4, 50 ml)
        Note: Need add 200 ml anhydrous ethanol before use.
      5. Eluate buffer (Reagent number: BG001-PC5, 100 ml)
    Tips：
    1. “*” For the first use, the magnetic beads must be thoroughly mixed ensuring the magnetic beads is fully dispersed.
    2. “**” Please add suitable anhydrous ethanol into washing buffer I and washing buffer II before use, and store at about 25 °C.
    3. Please prepare anhydrous ethanol by yourself.

  2. SARS-CoV-2 Nucleic Acid Detection Kit
    Notes:
    1. Store all the kit components at -15~-25 °C.
    2. Always check the expiration date prior to use. Do not use expired reagents.
    3. Protect SARS-CoV-2 reaction mix from light.
    4. RT-PCR Reaction Mix, RT-PCR Enzyme Mix, and SARS-CoV-2 reaction mix must be thawed and kept on cold block at all times during preparation and use.

    1. RT-PCR Reaction Mix (600 μl)
      Tris-HCl
      KCl
      MgCl2
      dNTPs
    2. RT-PCR Enzyme Mix (200 μl)
      DNA polymerase
      Reverse transcriptase
      RNase inhibitor
    3. SARS-CoV-2 reaction mix (200 μl)
      ORF1ab
      N gene
      RNase P specific Primers and probes
    4. Positive Control (500 μl)
      ORF1ab specific fragment pseudovirus particle
      N gene specific fragment pseudovirus particle
      RNase P virus-like particles
    5. Negative Control (500 μl)
      0.9% saline solution

Equipment

  1. Biological safety cabinet
  2. Refrigerated microcentrifuge
  3. Cold blocks
  4. Vortex mixer
  5. Dedicated adjustable pipettes
  6. Applied Biosystems 7500 Fast DX Real-Time PCR Instrument (Pub. No. 4406991)
  7. Magnetic separator

Procedure

Copyright: © 2020 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).

Category

Bioinformatics and Computational Biology

Systems Biology > Genomics > Phylogenetics

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