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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
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Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification.

Priyanka Valloly1, Rahul Roy1,2

  • 1Department of Chemical Engineering, Indian Institute of Science, Bangalore, Karnataka, India 560012.

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|September 28, 2022
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A new quantitative endpoint recombinase polymerase amplification (qeRPA) method estimates nucleic acid levels using final amplicon yield, avoiding complex equipment. This technique offers sensitive DNA detection and viral load estimation for field use in resource-limited settings.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Diagnostics

Background:

  • Quantitative polymerase chain reaction (qPCR) is accurate but requires thermal cyclers and real-time monitoring.
  • Existing isothermal methods also need sophisticated readers for continuous monitoring.
  • These limitations hinder the application of nucleic acid quantification in many settings.

Purpose of the Study:

  • To develop a semiquantitative isothermal method for nucleic acid quantification.
  • To adapt recombinase polymerase amplification (RPA) for endpoint analysis.
  • To enable nucleic acid detection without thermal cycling or real-time monitoring.

Main Methods:

  • Developed a phenomenological model for RPA dynamics.
  • Identified reaction conditions to constrain yield based on initial DNA concentration.
  • Validated predictions experimentally and termed the method quantitative endpoint RPA (qeRPA).

Main Results:

  • qeRPA demonstrated a correlation between amplicon yield and starting DNA concentration.
  • Achieved robust reduction in nonspecific amplification.
  • Detected DNA over five log orders with a detection limit of 100 molecules.
  • Estimated dengue virus viral load comparable to qPCR using a normalized endpoint intensity standard curve.

Conclusions:

  • qeRPA provides robust and sensitive nucleic acid estimation at near room temperature.
  • The method eliminates the need for real-time monitoring and thermal cyclers.
  • qeRPA is suitable for field deployment in resource-limited settings.