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Related Concept Videos

Real Time RT-PCR02:57

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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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Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
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Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR.

Lucien Jacky1, Dominic Yurk1,2, John Alvarado1

  • 1ChromaCode Inc., 2330 Faraday Ave Suite 100, Carlsbad, California 92008, United States.

Analytical Chemistry
|February 25, 2021
PubMed
Summary
This summary is machine-generated.

High-definition PCR (HDPCR) enables detecting up to 20 targets per well using standard qPCR instruments. This novel method increases diagnostic yield affordably without compromising throughput or cost for multiplex pathogen detection.

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Area of Science:

  • Molecular Diagnostics
  • Biotechnology
  • Infectious Disease Research

Background:

  • Quantitative polymerase chain reaction (qPCR) is the gold standard for molecular pathogen detection.
  • Current qPCR instruments typically detect only 4-6 analytes per sample, limiting multiplexing capabilities for complex diagnostic needs.
  • Clinical applications like simultaneous testing for SARS-CoV-2 and other respiratory pathogens necessitate higher multiplexing levels than standard qPCR offers.

Purpose of the Study:

  • To introduce and validate a novel method, high-definition PCR (HDPCR), for significantly increasing multiplexing capacity in standard qPCR.
  • To demonstrate that HDPCR can detect up to 20 targets per well without requiring new instrumentation or increasing costs.
  • To address the limitations of current qPCR for high-throughput screening scenarios demanding simultaneous detection of multiple pathogens.

Main Methods:

  • Developed and applied high-definition PCR (HDPCR), integrating TaqMan chemistry with robust encoding for enhanced multiplexing.
  • Utilized HDPCR with three distinct assays: a custom 20-Plex assay, an 8-Plex assay using predesigned single-plex assays, and a 9-Plex pathogen panel including SARS-CoV-2.
  • Tested the performance of HDPCR assays on various sample types using standard qPCR instrumentation and familiar workflows.

Main Results:

  • Successfully demonstrated HDPCR capability for detecting up to 20 targets per well.
  • Achieved high overall sample accuracies across all tested HDPCR assays: 98.8% for the 20-Plex, 98.3% for the 8-Plex, and 100% for the 9-Plex pathogen panel.
  • Validated HDPCR's effectiveness with SARS-CoV-2 and other common respiratory viruses.

Conclusions:

  • HDPCR technology significantly enhances the multiplexing capacity of existing qPCR instrumentation without additional cost or workflow changes.
  • This innovation meets the critical need for increased diagnostic yield in high-throughput screening, particularly for infectious disease panels.
  • HDPCR offers an affordable and efficient solution for mid-density multiplex diagnostics, leveraging the widespread install base of qPCR systems.