Precise Detection of Viral RNA by Programming Multiplex Rolling Circle Amplification and Strand Displacement

Area of Science:

• Molecular Biology
• Biotechnology
• Infectious Disease Diagnostics

Background:

• Accurate detection of viral infections like SARS-CoV-2 is crucial for public health.
• Existing laboratory-based PCR assays, while accurate, are not suitable for point-of-care settings.
• There is a need for rapid, sensitive, and specific point-of-care diagnostic tools.

Purpose of the Study:

• To develop a novel point-of-care method for ultraspecific and sensitive detection of SARS-CoV-2 RNA.
• To implement a multilayered specificity strategy to prevent false-positive results.
• To create a convenient visual detection assay comparable to qPCR.

Main Methods:

• Developed mCAD (multiplex rolling circle amplification and toehold-mediated strand displacement).
• Utilized sequence-specific nucleic acid hybridization across serial reaction steps for enhanced specificity.
• Integrated rolling circle amplification (RCA) for target amplification.

Main Results:

• mCAD demonstrated ultraspecific detection of SARS-CoV-2 RNA with high sensitivity.
• The method achieved performance comparable to quantitative PCR (qPCR).
• Visual detection of SARS-CoV-2 RNA was successfully demonstrated in both synthetic and clinical samples.

Conclusions:

• mCAD offers a highly specific, sensitive, and convenient point-of-care detection method for SARS-CoV-2 RNA.
• The multilayered specificity approach effectively eliminates false-positive detections.
• mCAD holds significant potential for broad clinical applications in nucleic acid detection beyond SARS-CoV-2.