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DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning

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Dynamic bidirectional diffusion-controlled multi-enzyme system for one-pot viral detection.

Jing Zhang¹, Xuening Shi¹, Yukun Ding¹

¹State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, School of Public Health, Jilin University, Changchun 130021, China.

Journal of Advanced Research

|July 7, 2026

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This study introduces a novel dual-phase system for rapid, sensitive virus detection using isothermal amplification and CRISPR-Cas12a technology. The new method overcomes previous limitations, offering highly accurate results in just 30 minutes.

Area of Science:

Biotechnology
Molecular Diagnostics
Point-of-Care Testing

Background:

Isothermal amplification and CRISPR-Cas detection offer rapid point-of-care virus diagnostics.
Conventional one-pot methods face mutual inhibition issues between amplification and CRISPR-Cas reactions, limiting performance.

Purpose of the Study:

To develop an integrated reaction system for efficient multi-enzyme coordination in a single tube.
To overcome inhibitory interactions in isothermal amplification and CRISPR-Cas detection.

Main Methods:

Designed a dynamic bidirectional diffusion-controlled RPA/CRISPR-Cas12a system using dual-phase separation.
Utilized glycerol for viscosity modulation and sucrose for density gradient creation, enabling spatial enzyme separation.
Integrated a 3D-printed nucleic acid extraction device for simplified sample preparation.

Keywords:

CRISPR-Cas12a Dynamic bidirectional diffusion Recombinase polymerase amplification Viral detection

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Main Results:

Achieved single-copy sensitivity with detection completed within 30 minutes.
Demonstrated over 100-fold higher sensitivity compared to conventional one-pot assays.
Validated the system for Norovirus detection in clinical and food samples.

Conclusions:

The dual-phase RPA/CRISPR-Cas12a system offers a simple, rapid, and highly sensitive nucleic acid detection platform.
The system is operationally simple and compatible with low-resource settings.
Highlights potential for decentralized and home-based virus diagnostics.