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

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Increasing SARS-CoV-2 RT-qPCR testing capacity by sample pooling.

Julia Alcoba-Florez¹, Helena Gil-Campesino¹, Diego García-Martínez de Artola¹

¹Servicio de Microbiología, Hospital Universitario N. S. de Candelaria, Santa Cruz de Tenerife, Spain.

International Journal of Infectious Diseases : IJID : Official Publication of the International Society for Infectious Diseases

|November 21, 2020

Summary

Pooling five samples for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) testing significantly increased capacity with minimal loss of sensitivity. This method enhances coronavirus disease 2019 (COVID-19) diagnostic capabilities.

Keywords:

COVID-19 SARS-CoV-2 Sample pooling Scalability Testing capacity

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Area of Science:

Molecular Biology
Virology
Public Health

Background:

Limited testing capacity for COVID-19 in Spain hindered outbreak control.
Effective strategies are needed to increase SARS-CoV-2 testing capacity.

Purpose of the Study:

To evaluate the use of sample pooling with RT-qPCR to enhance SARS-CoV-2 testing capacity.
To determine the optimal pool size for sensitive and reproducible COVID-19 diagnosis.

Main Methods:

Evaluated various pool sizes (5, 10, 15 samples) for RT-qPCR testing.
Assessed nasopharyngeal samples using pooled RT-qPCR in a tertiary hospital.
Compared two different RT-qPCR kits for pooled sample analysis.

Main Results:

A pool size of five samples demonstrated higher sensitivity than pools of 10 or 15.
The PathoFinder kit showed the lowest mean Ct shift between pooled and individual samples.
This strategy detected positive samples within a Ct range of 16.7-39.4.

Conclusions:

Pooling samples in groups of five for RT-qPCR increases SARS-CoV-2 testing capacity.
This approach offers a minimal loss of sensitivity compared to individual sample testing.