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This study introduces a novel nanopore sensing method for direct, label-free quantification of SARS-CoV-2 RNA in nasal swabs. This PCR-free approach offers accurate COVID-19 diagnosis, potentially detecting samples missed by traditional tests.

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Area of Science:

  • Biotechnology
  • Nanotechnology
  • Molecular Diagnostics

Background:

  • Current RNA-based COVID-19 diagnostics rely on enzymatic amplification, which can lead to inconsistent results due to arbitrary thresholding.
  • Accurate and sensitive detection of SARS-CoV-2 RNA is crucial for effective COVID-19 diagnosis and management.

Purpose of the Study:

  • To develop a PCR-free, label-free method for sensitive and quantitative detection of SARS-CoV-2 RNA in clinical samples.
  • To validate a novel solid-state nanopore sensing technology for COVID-19 diagnostics.
  • To assess the potential of nanopore sensing to identify samples that may evade detection by conventional methods.

Main Methods:

  • Utilized solid-state nanopore sensing for label-free quantification of SARS-CoV-2 RNA.
  • Employed a workflow involving reverse transcription, enzymatic digestion to dsDNA, and single-molecule counting.
  • Performed ratiometric quantification using dsDNA from a SARS-CoV-2 gene and a human reference gene.
  • Integrated sample preparation into a compact microfluidic device for semi-automated processing.

Main Results:

  • Demonstrated good correlation between the SARS-CoV-2 ratiometric nanopore index and RT-qPCR threshold cycle for positive samples.
  • Identified positive COVID-19 cases in clinical samples that were classified as negative by RT-qPCR.
  • Showcased the feasibility of a compact, semi-automated microfluidic device for rapid RNA quantification.

Conclusions:

  • Solid-state nanopore sensing provides a sensitive, label-free, and quantitative method for SARS-CoV-2 RNA detection.
  • This PCR-free approach offers a potential advantage in diagnosing COVID-19, including in samples missed by RT-qPCR.
  • The developed microfluidic system paves the way for automated, fast, and portable RNA quantification devices.