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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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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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Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression
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A Quantitative Real-Time PCR Assay for Measuring Poxvirus Replication and Cell Binding.

Brian M Ward1, Stephanie R Monticelli2,3

  • 1Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA. Brian_Ward@urmc.rochester.edu.

Methods in Molecular Biology (Clifton, N.J.)
|December 2, 2024
PubMed
Summary
This summary is machine-generated.

Quantitative real-time PCR (qPCR) offers a rapid and scalable method for quantifying viral genomes, outperforming traditional plaque assays. This study details a qPCR assay for vaccinia virus DNA and a cell-binding assay for assessing viral replication and infectivity.

Keywords:
Cell-binding assayGenome quantificationMonkeypox virusOrthopoxvirusQuantitative PCRVaccinia virusVirion quantification

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Area of Science:

  • Virology
  • Molecular Biology
  • Biotechnology

Background:

  • Quantitative real-time PCR (qPCR) is a valuable tool for viral genome quantification.
  • Traditional plaque assays for virus titration are time-consuming and less scalable.
  • Accurate measurement of viral replication and cell binding is crucial for virological studies.

Purpose of the Study:

  • To develop and describe a qPCR assay for quantifying vaccinia virus DNA.
  • To establish a sensitive cell-binding assay for evaluating virion-cell interactions.
  • To provide a robust methodology for comparative analysis of virus replication under various experimental conditions.

Main Methods:

  • Development of a quantitative real-time PCR (qPCR) assay targeting the vaccinia virus dsDNA genome.
  • Establishment of a straightforward cell-binding assay protocol using small concentrations of virions.
  • Application of qPCR for enumerating total viral genomes and assessing virus production.

Main Results:

  • The described qPCR assay accurately quantifies vaccinia virus genomes, serving as a surrogate for viral titration.
  • The cell-binding assay demonstrates sensitivity for detecting virion attachment, even with low inocula.
  • Both assays offer advantages in speed, linearity, and scalability compared to plaque assays.

Conclusions:

  • qPCR provides a fast, reliable, and scalable method for quantifying viral genomes and assessing virus replication.
  • The developed cell-binding assay is effective for evaluating mutations impacting virus production and infectivity.
  • These methodologies enhance the ability to study virus-host interactions and replication dynamics.