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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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On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

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Large-Scale RT-qPCR Diagnostics for Seed Potato Certification.

Olivier Schumpp¹, Amanda Bréchon^1,2, Justine Brodard¹

¹Plant Protection Department, Agroscope, 1260 Nyon, Switzerland.

Potato Research

|November 18, 2021

Summary

Agroscope now uses RT-qPCR for rapid virus detection in seed potatoes, replacing toxic Rindite. This method enhances quality control and aids in studying viral epidemiology and diversity in potato crops.

Keywords:

Certification Deep sequencing Molecular diagnostics Quality control Real-time RT-PCR Viral disease

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Area of Science:

Plant virology
Agricultural diagnostics
Molecular biology

Background:

Seed potato certification in Switzerland involves annual testing of approximately 300,000 tubers for regulated viruses.
Traditional methods required Rindite, a toxic compound, to break tuber dormancy for testing.
Since 2016, Agroscope has implemented RT-qPCR for direct analysis of dormant tubers.

Purpose of the Study:

To detail the implementation of RT-qPCR for routine virus detection in seed potato certification.
To leverage molecular data for quality control of diagnostic primers and probes.
To explore fundamental questions in viral strain epidemiology and discover novel virus species.

Main Methods:

Quantitative real-time PCR (RT-qPCR) applied directly to dormant seed potato tubers post-harvest.
Illumina sequencing for assessing primer/probe conformity against viral isolate sequences.
Data analysis for epidemiological insights and viral diversity assessment.

Main Results:

RT-qPCR provides rapid and reliable virus detection, eliminating the need for Rindite.
The method facilitates enhanced quality control of diagnostic reagents.
Generated datasets offer valuable information on viral epidemiology and potential discovery of new viruses.

Conclusions:

The adoption of RT-qPCR has modernized seed potato virus diagnostics in Switzerland, improving efficiency and safety.
This approach serves as a robust platform for ongoing quality assurance and scientific research into potato viruses.
The methodology supports both regulatory compliance and the advancement of knowledge in plant virology.