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Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

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Published on: December 23, 2020

Subgenomic RNA Detection in SARS-CoV-2 Assessing Replication and Inactivation Through Serial Passages, RT-qPCR, and

Talita da Silva França¹, Juliana Fernandes Amorim da Silva², Gabriella Christine Neves da Silva²

¹Virological Technology Laboratory, Bio-Manguinhos/FIOCRUZ, Rio de Janeiro 21040-900, Brazil.

International Journal of Molecular Sciences

|February 13, 2025

Summary

Subgenomic RNAs (sgRNAs) indicate active SARS-CoV-2 replication. Quantifying sgRNA and RNA intermediates using RT-qPCR successfully distinguished viable virus from inactivated samples, confirming inactivation after two passages.

Keywords:

RT-qPCR SARS-CoV-2 sgRNA

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Area of Science:

Virology
Molecular Biology
Infectious Diseases

Background:

Subgenomic RNAs (sgRNAs) are essential for SARS-CoV-2 protein synthesis and indicate active viral replication.
Distinguishing between active SARS-CoV-2 replication and non-viable viral particles is crucial for diagnostics and therapeutics.

Purpose of the Study:

To quantify sgRNA and negative RNA intermediates in SARS-CoV-2 samples.
To assess viral replication in virus samples inactivated by β-propiolactone (βPL).
To validate sgRNA as a marker for active SARS-CoV-2 replication.

Main Methods:

RT-qPCR was employed to quantify genomic and subgenomic RNAs (sgENV) and RNA intermediates (ENV-).
Inactivated SARS-CoV-2 samples underwent five blind serial passages (BSs).
Transmission electron microscopy was used to visualize viral particles.

Main Results:

Positive controls maintained consistent viral titers across passages.
sgENV was undetectable after the second passage in inactivated samples, indicating successful inactivation.
Replication kinetics showed distinct profiles for active versus inactivated virus, with sgRNA absence correlating with inactivation.

Conclusions:

sgRNA serves as a reliable marker for active SARS-CoV-2 replication.
RT-qPCR quantification of sgRNA and RNA intermediates can differentiate viable from non-viable virus.
These findings support the development of diagnostic and therapeutic strategies targeting viral replication.