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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Point-of-Care Diagnostics

Background:

  • Quantitative nucleic acid amplification tests (qNAATs) are crucial for managing infectious diseases, including HIV and SARS-CoV-2.
  • Current gold standard quantitative PCR (qPCR) requires laboratory infrastructure, limiting point-of-care (POC) applications.
  • Isothermal amplification methods offer a promising alternative for developing portable NAATs.

Purpose of the Study:

  • To develop a rapid, POC-compatible qNAAT method for infectious disease quantification.
  • To explore the potential of discrete fluorescent signals from isothermal amplification for quantitative analysis.
  • To establish a novel method for quantifying nucleic acids using recombinase polymerase amplification (RPA) nucleation sites.

Main Methods:

  • Utilized recombinase polymerase amplification (RPA) on paper membranes to generate discrete fluorescent nucleation sites.
  • Developed an image-analysis algorithm to count nucleation sites for quantification.
  • Integrated a mobile phone-based system for image capture and onboard data processing.

Main Results:

  • Demonstrated that the number of RPA nucleation sites directly correlates with the input quantity of target nucleic acids.
  • Successfully quantified HIV-1 DNA and viral RNA within 20 minutes.
  • Achieved accurate quantification across a range of 67-3000 nucleic acid copies per reaction.
  • Validated a mobile phone-based system for POC qNAATs with minimal instrumentation.

Conclusions:

  • The number of discrete fluorescent nucleation sites in RPA reactions can be effectively used for nucleic acid quantification.
  • This method provides a rapid and sensitive approach for point-of-care quantitative nucleic acid amplification tests.
  • The developed mobile phone-based system shows significant potential for decentralized infectious disease diagnostics.