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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

58.0K
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...
58.0K

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Related Experiment Video

Updated: Aug 22, 2025

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
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Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

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Development and validation of RT-PCR assays for testing for SARS-CoV-2.

Kanti Pabbaraju1, Anita A Wong1, Mark Douesnard1

  • 1Alberta Precision Laboratories, Public Health Laboratory, Calgary, Alberta, Canada.

Journal of the Association of Medical Microbiology and Infectious Disease Canada = Journal Officiel De L'Association Pour La Microbiologie Medicale Et L'Infectiologie Canada
|November 7, 2022
PubMed
Summary

Rapid development of real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enabled early public health response. These validated assays proved crucial for pandemic containment.

Keywords:
CoV-2SARSassaysdevelopmentemerging or re-emerging diseaseslaboratorymolecular methodsvalidations

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Area of Science:

  • Virology
  • Molecular Biology
  • Public Health

Background:

  • The emergence of SARS-CoV-2 necessitated rapid development of diagnostic assays.
  • Lack of commercial assays highlighted the need for laboratory-developed tests for clinical diagnosis and public health.
  • Timely detection is critical for managing emerging infectious diseases.

Purpose of the Study:

  • To outline the development and validation of laboratory-developed reverse-transcriptase polymerase chain reaction (RT-PCR) assays for SARS-CoV-2.
  • To describe the progression of assay development to meet increasing testing demands.
  • To ensure sensitive and specific detection of SARS-CoV-2 for public health response.

Main Methods:

  • Development and validation of SARS-CoV-2-specific and pan-coronavirus gel-based assays.
  • Transition to real-time RT-PCR assays targeting envelope and RNA-dependent RNA polymerase genes.
  • Implementation of multiplexing and internal extraction controls to enhance throughput.

Main Results:

  • Developed real-time RT-PCR assays demonstrated comparable sensitivity and specificity to WHO and CDC recommended assays.
  • Assay progression successfully accommodated high anticipated sample volumes.
  • Multiplexing and internal controls improved testing efficiency.

Conclusions:

  • Early availability of validated real-time RT-PCR assays was essential for pandemic response.
  • Laboratory-developed assays provided critical time for public health authorities to contain transmission.
  • Preparedness through rapid assay development is key to managing public health crises.