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Related Concept Videos

Real Time RT-PCR02:57

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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Virus Propagation and Cell-Based Colorimetric Quantification.

Jia-Yi Tan1, Jo-Ern Wong1, Nurhafiza Zainal2

  • 1Tropical Infectious Diseases Research and Education Centre (TIDREC), Higher Institution Center of Excellence (HICoE), Universiti Malaya.

Journal of Visualized Experiments : Jove
|April 24, 2023
PubMed
Summary
This summary is machine-generated.

This study details a reliable focus forming assay (FFA) protocol for quantifying Zika virus (ZIKV) and other viruses. The optimized method, suitable for high-throughput screening, aids in developing ZIKV vaccines and treatments.

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Area of Science:

  • Virology
  • Immunology
  • Cell Biology

Background:

  • Zika virus (ZIKV) is a significant public health concern, linked to severe congenital abnormalities and neurological disorders.
  • Accurate virus quantification is essential for understanding ZIKV pathogenesis and developing effective countermeasures, including vaccines and therapeutics.
  • Existing virus quantification methods may have limitations, necessitating refined protocols for broader applicability.

Purpose of the Study:

  • To describe and optimize a focus forming assay (FFA) protocol for propagating and quantifying Zika virus (ZIKV).
  • To adapt the FFA for both standard (24-well) and high-throughput (96-well) formats.
  • To provide a versatile protocol applicable to various viruses, including those that do not form plaques.

Main Methods:

  • ZIKV propagation in Vero cells over 3 days, followed by supernatant harvesting.
  • Quantification using FFA involving inoculation onto Vero cells, incubation, and optimized staining (fixation, permeabilization, blocking, antibody binding, substrate incubation).
  • Foci visualization and counting via stereo microscopy (manual) or software analysis (automated).

Main Results:

  • A reproducible and relatively rapid (3-4 days) protocol for ZIKV quantification using FFA.
  • Demonstrated optimization of foci size for improved assay performance.
  • Successful adaptation of the FFA for both 24-well and 96-well formats, enabling manual and automated counting.

Conclusions:

  • The described FFA protocol offers a robust method for ZIKV quantification and research.
  • The optimized protocol is adaptable for various viruses, including non-plaque-forming types.
  • This assay serves as a valuable tool for ZIKV research and the detection of other clinically relevant viruses.