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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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Updated: Jan 16, 2026

Remote Laboratory Management: Respiratory Virus Diagnostics

Remote Laboratory Management: Respiratory Virus Diagnostics

Published on: April 6, 2019

Comparative Performance of Digital PCR and Real-Time RT-PCR in Respiratory Virus Diagnostics.

Irene Bianconi¹, Giovanna Viviana Pellecchia², Elisabetta Maria Incrocci¹

¹Laboratory of Microbiology and Virology, Hospital of Bolzano (SABES-ASDAA), Teaching Hospital of Paracelsus Medical University (PMU), 39100 Bolzano-Bozen, BZ, Italy.

|September 27, 2025

Summary

Digital PCR (dPCR) offers more accurate quantification of respiratory viruses like influenza and SARS-CoV-2 compared to Real-Time RT-PCR, especially for high viral loads. This improved accuracy aids in understanding viral co-infections during the tripledemic.

Keywords:

Real-Time RT-PCR digital PCR respiratory viruses tripledemic virus diagnostics

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Last Updated: Jan 16, 2026

Remote Laboratory Management: Respiratory Virus Diagnostics

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Published on: April 6, 2019

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Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Published on: March 31, 2021

Area of Science:

Molecular Diagnostics
Virology
Infectious Diseases

Background:

Respiratory viral infections represent a significant global health challenge.
Molecular diagnostics like Real-Time RT-PCR are crucial but face challenges in precise viral RNA quantification.
Digital PCR (dPCR) offers absolute quantification, potentially enhancing diagnostic accuracy for co-infections.

Purpose of the Study:

To compare the diagnostic accuracy of dPCR and Real-Time RT-PCR for quantifying common respiratory viruses.
To evaluate viral load quantification across different categories (high, medium, low) during the 2023-2024 tripledemic.
To assess the performance of dPCR and Real-Time RT-PCR in detecting influenza A, influenza B, RSV, and SARS-CoV-2.

Main Methods:

Analysis of 123 respiratory samples using both dPCR and Real-Time RT-PCR.
Stratification of samples into high, medium, and low viral load categories based on cycle threshold (Ct) values.
Comparative quantification of viral loads for influenza A, influenza B, RSV, and SARS-CoV-2.

Main Results:

dPCR showed superior accuracy in quantifying high viral loads of influenza A, influenza B, and SARS-CoV-2.
dPCR demonstrated greater accuracy for medium viral loads of RSV.
dPCR provided enhanced consistency and precision, particularly for intermediate viral levels, compared to Real-Time RT-PCR.

Conclusions:

dPCR holds significant potential for improving respiratory virus diagnostics and understanding co-infection dynamics.
The study highlights dPCR's advantages in accuracy and precision for key respiratory pathogens.
Current limitations for routine dPCR implementation include higher costs and lower automation compared to Real-Time RT-PCR.