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Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

Published on: November 1, 2018

Framework for using quantitative PCR as a nonculture based method to estimate virus infectivity.

Brian M Pecson¹, Martin Ackermann, Tamar Kohn

¹Laboratory of Environmental Chemistry, School of Architecture, Civil and Environmental Engineering, École Polytechnique Fédérale de Lausanne, Station 2, 1015 Lausanne, Switzerland.

Environmental Science & Technology

|February 17, 2011

Summary

This study presents a new theoretical framework to accurately measure virus disinfection efficiency using quantitative PCR (qPCR). The developed model links genome damage detected by qPCR to actual viral infectivity, overcoming previous limitations.

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Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

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Published on: November 1, 2018

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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

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Published on: March 30, 2015

Area of Science:

Virology
Molecular Biology
Environmental Science

Background:

Quantitative PCR (qPCR) is widely used to measure virus disinfection efficacy.
qPCR measurements can be criticized for potential false-positive signals and may not directly correlate with viral infectivity.
Existing methods often overlook that qPCR targets only a fraction of the viral genome, complicating direct infectivity comparisons.

Purpose of the Study:

To develop a theoretical framework that accurately relates viral infectivity to genome damage measured by qPCR.
To establish a method for quantifying genome-wide damage based on qPCR amplification of specific genomic regions.
To validate the framework using a model virus and assess its applicability to nonculturable viruses.

Main Methods:

Developed a theoretical model assuming single-hit inactivation and Poissonian damage distribution.
Quantified genome damage by analyzing qPCR amplification of smaller genome sections.
Utilized UV(254) inactivation studies with bacteriophage MS2 for experimental validation.
Modified the framework to account for heterogeneous UV sensitivity across the genome.

Main Results:

The developed framework accurately estimates viral infectivity based on qPCR-derived genome damage.
UV(254) inactivation of MS2 revealed heterogeneous sensitivity of genome regions to UV damage.
The modified framework successfully accounted for deviations from the Poissonian damage assumption.
The framework demonstrated the potential for monitoring infectivity of nonculturable viruses like norovirus.

Conclusions:

The novel theoretical framework provides a more accurate method for assessing virus disinfection efficiency using qPCR.
The approach accounts for genome damage across the entire viral genome, not just targeted regions.
This method has significant implications for public health, particularly for monitoring nonculturable pathogens and ensuring disinfection efficacy.