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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: Oct 12, 2025

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

An Ultrafast One-Step Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Detection of SARS-CoV-2.

Jadranka Milosevic^1,2, Mengrou Lu², Wallace Greene³

¹Captis Diagnostics Inc., Pittsburgh, PA, United States.

Frontiers in Microbiology

|November 22, 2021

Summary

We created a rapid, one-step RT-qPCR test for SARS-CoV-2 detection, delivering results in 30 minutes. This ultrafast assay achieves 100% clinical sensitivity and specificity, significantly improving diagnostic speed.

Keywords:

COVID-19 SARS-CoV-2 detection nasopharyngeal swab one-step RT-qPCR assay ultrafast

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Last Updated: Oct 12, 2025

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

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Published on: March 31, 2021

Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP

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Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses

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Published on: November 10, 2023

Area of Science:

Molecular Biology
Virology
Diagnostic Assays

Background:

Conventional RT-qPCR assays for SARS-CoV-2 detection are time-consuming.
There is a need for faster diagnostic methods to enable rapid public health responses.

Purpose of the Study:

To develop and validate an ultrafast, one-step RT-qPCR assay for the rapid detection of SARS-CoV-2.
To significantly reduce the turnaround time for molecular diagnostic testing of COVID-19.

Main Methods:

Development of a one-step RT-qPCR assay integrating reverse transcription and quantitative polymerase chain reaction.
Optimization of reaction times for RT (1 min) and PCR cycles (1s denaturation, 1s extension).
Validation using a cohort of 60 nasopharyngeal swab samples on a Bio-Rad CFX96 instrument.

Main Results:

The ultrafast RT-qPCR assay successfully detected SARS-CoV-2 in 30 minutes.
Significant reductions in reaction times were achieved compared to conventional methods.
The assay demonstrated 100% clinical sensitivity and 100% clinical specificity in the tested sample cohort.

Conclusions:

The developed one-step RT-qPCR assay provides a highly sensitive and specific method for rapid SARS-CoV-2 detection.
This assay offers a substantial improvement in diagnostic speed, facilitating timely clinical and public health decisions.
The ultrafast assay is suitable for benchtop use, enhancing accessibility for rapid molecular diagnostics.