Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Laboratory-based diagnostics like RT-qPCR are crucial but limited in resource-constrained settings during public health emergencies.
• There is a need for accurate, rapid, and accessible diagnostic methods to increase testing capacity and improve disease management.
• CRISPR-Cas9 and lateral flow immunochromatography offer promising avenues for developing novel diagnostic platforms.

Purpose of the Study:

• To develop an original nucleic acid detection platform combining CRISPR-Cas9 and lateral flow immunochromatography.
• To create a rapid, sensitive, and accessible diagnostic tool for point-of-care testing.
• To validate the platform's efficacy for detecting viral nucleic acids, including multiplexed detection.

Main Methods:

• Leveraged CRISPR-Cas9 and lateral flow immunochromatography with isothermal amplification and biotinylated primers.
• Utilized the interaction between CRISPR-Cas9 R-loops and fluorescein-labeled probes for visual readout on lateral flow devices.
• Validated using clinical samples of SARS-CoV-2 and spiked samples for multiplexed detection of SARS-CoV-2 and Influenza A (H1N1).

Main Results:

• Achieved rapid (1 hour) and sensitive nucleic acid detection with a limit of 1-10 copies/μL at low temperatures.
• Demonstrated a robust signal-to-noise ratio and utilized accessible molecular elements compared to other assays.
• Successfully performed multiplexed detection of SARS-CoV-2 and Influenza A (H1N1) in spiked samples.

Conclusions:

• The developed platform is a versatile and manageable tool for point-of-care testing.
• It enhances patient outcomes and strengthens pandemic preparedness through accessible diagnostics.
• The method holds significant potential for multiplexed diagnostics of respiratory viruses.