PCR Diagnostics for Novel Coronavirus

Oct 25, 2024

Reverse Transcriptase PCR for Diagnosing 2019 Novel Coronavirus

Introduction

  • Presented by Lindsay Alton, Postdoctoral Research Associate, University College London, Pandora ID net consortium.
  • Purpose: Method for diagnosing the 2019 novel coronavirus (2019-nCoV) using reverse transcriptase PCR (RT-PCR).

Safety Protocols

  • Biosafety Level 3 (BSL-3) conditions required due to transmission risk.
    • Use Class 1 or Class 2 microbiological safety cabinet.
    • Follow local BSL-3 health and safety rules.
  • Maintain a clean chain of laboratories to prevent sample contamination.

RNA Extraction Method

  • Kit Used: Direct Sole RNA Miniprep Kit.
  • Sample Preparation:
    • Use 80 µL of sample + 240 µL of Trizol mix, mix for 5 minutes.
    • Add equal volume of 95-100% ethanol and mix thoroughly.
    • Transfer to a spin column in a collection tube.
    • Disinfect outside of tubes and spin column before centrifuging.
    • Centrifuge at 10,000-16,000 G for 30 seconds.
    • Repeat washing steps with RNA wash buffer twice.
    • Optional: DNAse treatment recommended.
  • RNA Elution:
    • Add 50 µL of RNAse-free water or 25 µL for higher concentration.
    • Store eluted RNA at -70°C.

Ensuring Extraction Quality

  • Check extraction efficacy by quantifying eluted RNA (methods in accompanying PDF).

PCR Preparation

  • PCR Setup:
    • Separate areas for PCR preparation and thermocycling to prevent contamination.
  • Master Mix Components:
    • Nuclease-free water, 2X RT-PCR buffer, forward and reverse primers + probe for 2019-nCoV, and 25X RT-PCR enzyme mix.
  • Primer Handling:
    • Order specific primers, reconstitute in nuclease-free water, aliquot into tubes for storage.

Master Mix Creation

  • Master mix should include reagents in an RNAse-free tube.
  • Example setup: 3 patient samples, 3 repetitions each, plus positive and negative controls.
  • Mix the master mix thoroughly before aliquoting into PCR tubes.

PCR Amplification

  • Follow specific instructions for the thermocycler used (e.g., Corbett Research Rotor Gene).
  • Cycle Settings:
    • 50°C for 15 minutes, 95°C for 3 minutes, followed by 45 cycles of:
      • 95°C for 15 seconds
      • 60°C for 30 seconds
  • Ensure fluorescence acquisition in the correct channel (e.g., SCI 5).

Result Analysis

  • PCR results analyzed using PCR cycle curves.
    • Set threshold for detection above background levels (negative controls).
  • Note: This PCR is qualitative, not quantitative; it only identifies the presence of 2019-nCoV RNA.
  • For validation, positive samples should be sequenced if a positive control is unavailable.

Additional Resources

  • For more information on diagnosing 2019-nCoV, consult:
    • Pandora ID net
    • Global Health Network's 2019-nCoV Knowledge Hub
    • WHO website (all referenced in accompanying PDF)