Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification.

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.