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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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Updated: May 18, 2026

Plaquing of Herpes Simplex Viruses

Plaquing of Herpes Simplex Viruses

Published on: November 5, 2021

Rapid susceptibility testing for herpes simplex virus type 1 using real-time PCR.

Martha T van der Beek¹, Eric C J Claas, Caroline S van der Blij-de Brouwer

¹Department of Medical Microbiology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands. m.t.van_der_beek@lumc.nl

Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology

|October 3, 2012

Summary

A new DNA reduction assay (DRA) offers a rapid and accurate method for testing herpes simplex virus type 1 (HSV-1) susceptibility to antiviral drugs. This PCR-based test provides reliable results comparable to traditional methods.

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Published on: November 27, 2019

Area of Science:

Virology
Molecular Biology
Clinical Diagnostics

Background:

Traditional herpes simplex virus type 1 (HSV-1) susceptibility testing relies on plaque reduction assays (PRA), which are laborious, time-consuming, and require manual interpretation.
There is a need for more efficient and objective methods for HSV-1 antiviral susceptibility testing in clinical settings.

Purpose of the Study:

To develop and validate an internally controlled, real-time PCR-based phenotypic susceptibility test for HSV-1.
The goal was to create a test suitable for clinical diagnostic use, offering improved efficiency and accuracy.

Main Methods:

A DNA reduction assay (DRA) was developed, involving a 48-hour pre-culture followed by a 24-hour culture with antivirals.
Viral DNA concentration was quantified using an internally controlled real-time PCR, with normalization for cell count and lysis via beta-globin PCR.
The DRA was validated against 26 HSV-1 isolates with known susceptibility to aciclovir and foscarnet, comparing results with PRA and sequence analysis.

Main Results:

The DRA demonstrated results consistent with PRA for both aciclovir and foscarnet susceptibility.
The assay showed good discriminative value for detecting low-level antiviral resistance associated with UL30 gene mutations.
A 48-hour pre-culture period was sufficient to obtain results within a clinically relevant 5-day timeframe.

Conclusions:

The DNA reduction assay (DRA) is an accurate, rapid, and user-friendly phenotypic susceptibility test for HSV-1.
This method provides a valuable alternative to traditional assays for clinical diagnostics, enabling timely and reliable antiviral susceptibility determination.