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

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Published on: March 31, 2021

RT-qPCR Diagnostics: The "Drosten" SARS-CoV-2 Assay Paradigm.

Stephen Bustin¹, Sara Kirvell¹, Jim F Huggett²

¹Medical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University Chelmsford, Chelmsford CM1 1SQ, UK.

International Journal of Molecular Sciences

|August 27, 2021

Summary

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a robust tool for detecting RNA viruses like SARS-CoV-2. While specific assays may have limitations, the overall RT-qPCR methodology ensures reliable COVID-19 testing.

Keywords:

COVID-19 SARS-CoV-2 molecular diagnosis qPCR reverse transcription

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Last Updated: Oct 22, 2025

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Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses

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Area of Science:

Molecular Biology
Virology
Diagnostic Technologies

Background:

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a key diagnostic method for RNA pathogens.
The COVID-19 pandemic highlighted RT-qPCR's role in rapid, sensitive mass testing for SARS-CoV-2.
Specific RT-qPCR assays targeting viral genes have faced scrutiny regarding their scientific validity.

Purpose of the Study:

To analyze a specific RT-qPCR assay targeting the RNA-dependent RNA polymerase gene of SARS-CoV-2.
To evaluate the criticisms regarding molecular and methodological flaws in this assay.
To assess the overall robustness and validity of RT-qPCR for COVID-19 detection.

Main Methods:

Detailed analysis of a specific RT-qPCR assay for SARS-CoV-2 detection.
Evaluation of primer and probe sequences for complementarity to target genes.
Assessment of potential impact of sequence errors on assay sensitivity and specificity.

Main Results:

The study identified some limitations within the analyzed RT-qPCR assay.
However, the findings underscore the general robustness of the RT-qPCR methodology for SARS-CoV-2 detection.
Sequence errors, if present, may reduce analytical sensitivity but are unlikely to cause significant false-positive results.

Conclusions:

RT-qPCR remains a reliable method for SARS-CoV-2 diagnosis.
While assay-specific limitations exist, they do not invalidate the overall technique.
Quality assurance measures in diagnostic laboratories help mitigate potential issues and ensure accurate testing.