RT-qPCR for diagnosing COVID-19 (former 2019-nCoV)

Summary

In this informative video, Lindsay Alton from University College London demonstrates the reverse transcriptase PCR method for diagnosing COVID-19 using viral RNA. She emphasizes strict biosafety protocols and the importance of maintaining a contamination-free laboratory environment. The process includes RNA extraction using a specialized kit, preparation of PCR reagents, and careful setup of the thermal cycler with specific cycling conditions. The video also covers data analysis techniques for interpreting PCR results, ensuring reliable detection of the virus. Comprehensive guidelines and additional resources are provided for further reference.

Takeaways

• 😀 RT-PCR is a method used to diagnose the 2019 novel coronavirus (COVID-19) by detecting its RNA.
• 🔬 Biosafety Level 3 (BSL-3) precautions are essential when handling samples to prevent contamination and transmission.
• 🧪 A Direct Sol RNA MiniPrep kit is recommended for RNA extraction from samples.
• 🧼 All equipment and surfaces should be disinfected before and after sample handling to contain aerosols.
• 🔄 The RNA extraction involves mixing samples with lysis buffer and ethanol, followed by centrifugation.
• 📋 A clean chain of laboratories is necessary to avoid cross-contamination during PCR preparation and amplification.
• 🔧 A one-step real-time RT-PCR approach is used to synthesize complementary DNA (cDNA) and conduct PCR in the same tube.
• 💧 Specific primers and probes for the 2019-nCoV need to be ordered and prepared before starting the PCR process.
• 📊 After PCR amplification, results can be analyzed to determine the presence of the virus's RNA in the samples.
• 🌐 Additional resources and guidelines for COVID-19 diagnosis can be found in the accompanying PDF and through health organizations.

Q & A

What is the main purpose of the video?

The video demonstrates the reverse transcriptase PCR method used to diagnose the 2019 novel coronavirus (2019-nCoV) by utilizing the virus's nucleic acid.

What are the biosafety requirements for handling 2019-nCoV?

BSL-3 conditions must be followed, including using a class I or II microbiological safety cabinet due to the potential for human-to-human transmission of the virus.

What is the first step in the RNA extraction process?

The first step involves adding 80 µL of the sample to 240 µL of Trizol, mixing thoroughly for 5 minutes, and then adding an equal volume of ethanol.

What is the significance of using a clean chain of laboratories?

A clean chain of laboratories prevents cross-contamination between PCR samples, ensuring accurate and reliable results.

What reagents are needed to prepare the PCR master mix?

The PCR master mix requires nuclease-free water, reverse transcriptase PCR buffer, specific primers and probes for 2019-nCoV, and a reverse transcriptase enzyme mix.

How should RNA samples be labeled during the PCR process?

RNA samples should be clearly labeled to keep track of the order in which they are added to the PCR tubes.

What are the thermocycler conditions for the PCR amplification?

The conditions include holding at 50°C for 15 minutes, 95°C for 3 minutes, and then 45 cycles of 95°C for 15 seconds and 60°C for 30 seconds.

Why is it recommended to aliquot stock solutions of primers?

Aliquoting stock solutions minimizes freeze-thaw cycles and prevents contamination, preserving the integrity of the reagents.

What should be done if a positive control for 2019-nCoV is not available?

If a positive control is not available, it is important to sequence positive samples to validate the PCR results.

Where can additional resources and information about diagnosing 2019-nCoV be found?

Additional resources can be found through the Pandora ID Net, Global Health Networks 2019-nCoV knowledge hub, and the WHO's website, as referenced in the accompanying protocol PDF.