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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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Related Experiment Video

Updated: Nov 10, 2025

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

Published on: June 23, 2022

Adjusting the Cut-Off and Maximum Pool Size in RT-qPCR Pool Testing for SARS-CoV-2.

Murilo S Costa¹, Hugo I Sato², Raissa P Rocha²

¹Graduate Program in Infectology and Tropical Medicine, Medical School, Universidade Federal de Minas Gerais, Belo Horizonte 30130-100, MG, Brazil.

|April 3, 2021

Summary

This summary is machine-generated.

This study introduces a new method for adjusting cycle threshold (Ct) values in pooled RT-qPCR testing for SARS-CoV-2. This ensures accurate COVID-19 detection even with diluted samples, enhancing large-scale testing consistency.

Keywords:

COVID-19 RT-qPCR SARS-CoV-2 pool testing

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Area of Science:

Molecular Biology
Infectious Disease Diagnostics
Public Health

Background:

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is crucial for detecting SARS-CoV-2 RNA and monitoring COVID-19.
Pool testing in RT-qPCR can increase testing efficiency but introduces challenges due to sample dilution.
Maintaining test sensitivity and accuracy in pooled samples is essential for reliable COVID-19 surveillance.

Purpose of the Study:

To propose a methodology for determining maximum pool sizes and adjusting cycle threshold (Ct) values in RT-qPCR pool testing.
To ensure that samples detectable in individual testing remain detectable when tested in pools.
To enhance the consistency and reliability of RT-qPCR pool testing for large-scale COVID-19 detection.

Main Methods:

Developed a procedure to calculate maximum pool size and adjust Ct cut-off values to compensate for dilution in pooled RT-qPCR.
Explicitly defined the trade-off between pool size and test sensitivity.
Validated the methodology through pool testing of adults with flu-like symptoms at a COVID-19 reference unit.

Main Results:

The proposed methodology successfully compensated for dilution effects in pooled RT-qPCR testing.
Samples near the limit of detection in individual tests were accurately identified in pooled tests.
The adjusted cut-off values, dependent on pool size, maintained detection sensitivity and avoided false negatives.

Conclusions:

The developed methodology ensures compatible detectability scales between individual and pooled RT-qPCR sample processing.
This approach enhances the consistency of RT-qPCR pool testing, making it more reliable for large-scale COVID-19 surveillance.
The findings support the increased use of RT-qPCR pool testing to improve the efficiency and reach of COVID-19 diagnostics.