A toehold-triggered switchable three-way junction protective nanoprobe for RNase H-assisted HBV rcDNA detection

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Nanotechnology

Background:

• Chronic hepatitis B virus (HBV) infection is a major global health concern.
• Accurate quantification of HBV relaxed circular DNA (rcDNA) is crucial for disease management.
• Existing amplification methods often lack specificity, hindering accurate viral load assessment.

Purpose of the Study:

• To develop a highly specific and sensitive diagnostic platform for HBV rcDNA quantification.
• To overcome the limitations of conventional isothermal amplification techniques.
• To engineer a novel DNA nanoprobe for improved viral detection.

Main Methods:

• Designed a toehold-triggered switchable DNA three-way junction (3WJ) protective nanoprobe.
• Integrated RNase H-assisted target recycling amplification (RATRA) with toehold-mediated strand displacement (TMSD).
• Utilized a dual-verification mechanism for enhanced specificity and a nanoscale cavity for steric hindrance.

Main Results:

• Achieved a limit of detection (LOD) of 87.28 fM with a broad linear dynamic range.
• Demonstrated exceptional specificity, capable of discriminating single-base mutations.
• Clinical serum sample validation showed strong concordance with quantitative real-time PCR (qRT-PCR).

Conclusions:

• The developed nanoprobe platform offers a robust, modular, and cost-effective solution for HBV diagnostics.
• This technology holds promise for point-of-care viral detection and monitoring.
• The study presents a significant advancement in sensitive and specific nucleic acid detection.