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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

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

Published on: March 31, 2021

A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection.

Martin A M Reijns¹, Louise Thompson², Juan Carlos Acosta³

¹MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, United Kingdom.

|December 15, 2020

Summary

A new, affordable multiplex assay for detecting SARS-CoV-2 (the virus that causes COVID-19) offers comparable performance to commercial tests. This cost-effective method, utilizing quantitative reverse transcription PCR (RT-qPCR), can improve accessibility for widespread diagnostic testing.

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Area of Science:

Molecular Biology
Virology
Infectious Diseases

Background:

The COVID-19 pandemic necessitates sensitive, specific, and affordable diagnostic tests for SARS-CoV-2.
Current RT-qPCR assays are sensitive but often expensive and face supply chain limitations.
There is a need for cost-effective diagnostic solutions, especially for low-resource settings.

Purpose of the Study:

To develop and validate a multiplex RT-qPCR assay for SARS-CoV-2 detection.
To assess the assay's performance against commercial standards.
To evaluate the assay's cost-effectiveness and potential for high-volume testing.

Main Methods:

Development of a multiplex assay targeting SARS-CoV-2, including human (RPP30) and viral (PhHV-1) controls.
Validation of the assay's performance compared to existing commercial kits.
Analysis of sample variability and correlation between human and viral nucleic acid levels.

Main Results:

The developed multiplex assay demonstrated performance comparable to commercial RT-qPCR kits.
The assay achieved comparable results at a significantly reduced cost (approximately 10% of commercial assays).
A statistically significant correlation was found between human nucleic acid levels and SARS-CoV-2 detection, highlighting the utility of the human control.

Conclusions:

A robust and cost-effective multiplex RT-qPCR assay for SARS-CoV-2 has been established.
The assay's affordability and inclusion of controls can enhance diagnostic capacity, particularly in resource-limited regions.
The human control provides a quantitative measure of sample quality, potentially reducing false-negative rates.