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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.
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In 1971, Peter Perlman and Eva Engvall developed an Enzyme-linked immunosorbent assay (ELISA or EIA). ELISA differs from western blot in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane.
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Related Experiment Video

Updated: Oct 31, 2025

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1
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Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

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A Semi-Quantitative Isothermal Diagnostic Assay Utilizing Competitive Amplification.

Christopher P Mancuso1, Zhi-Xiang Lu1,2, Jason Qian1,2,3

  • 1Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.

Analytical Chemistry
|June 28, 2021
PubMed
Summary

A new quantitative recombinase polymerase amplification (qRPA) test offers rapid, semi-quantitative results for infectious diseases like HIV and COVID-19. This field-deployable molecular diagnostic is robust and suitable for at-home viral load monitoring.

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Last Updated: Oct 31, 2025

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Area of Science:

  • Molecular diagnostics
  • Infectious disease detection
  • Nucleic acid amplification

Background:

  • Current diagnostic methods for infectious diseases are either slow and expensive or rapid but provide only binary results.
  • There is a critical need for rapid, inexpensive, sensitive, and field-deployable quantitative diagnostic tools.
  • Existing rapid assays lack the ability to measure analyte concentration, hindering effective disease management.

Purpose of the Study:

  • To develop a novel molecular diagnostic test, quantitative recombinase polymerase amplification (qRPA), for semi-quantitative nucleic acid detection.
  • To evaluate the performance of qRPA in quantifying DNA, RNA, and viral titers in patient samples.
  • To assess the robustness of qRPA compared to traditional recombinase polymerase amplification (RPA) under environmental perturbations.

Main Methods:

  • Development of a qRPA assay incorporating competitive amplification within a standard RPA framework.
  • Application of qRPA to quantify nucleic acids from HIV and COVID-19 patient samples.
  • Testing the robustness of qRPA against environmental variations compared to RPA.

Main Results:

  • The qRPA assay successfully provided semi-quantitative information on target nucleic acids.
  • qRPA demonstrated the ability to quantify DNA, RNA, and viral titers in clinical samples from HIV and COVID-19 patients.
  • The developed qRPA assay exhibited enhanced robustness to environmental changes compared to conventional RPA.

Conclusions:

  • qRPA is a promising molecular diagnostic tool capable of semi-quantitative nucleic acid detection.
  • The assay's ability to quantify viral loads in patient samples supports its utility in managing infectious diseases.
  • qRPA's robustness and potential for field-deployability make it suitable for applications such as at-home monitoring of viral infections.