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Updated: Sep 10, 2025
DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning
Published on: May 10, 2024
Sample-to-answer nucleic acid detection using a fully integrated microdevice for nucleic acid extraction and
Shuhao Zhao1, Yunyun Zhang1, Yifei Wang2
1School of Instrument Science and Technology, State Key Laboratory for Manufacturing System Engineering, Xi'an Jiaotong University, Xi'an, 710049, Shaanxi, China; State Industry-Education Integration Center for Medical Innovations, Xi'an Jiaotong University, Xi'an, 710054, Shaanxi, China; Key Laboratory of Advanced Biological Detection Technologies and Instruments of Shaanxi Province, Xi'an Jiaotong University, Xi'an, 710054, Shaanxi, China.
This study introduces a novel one-pot digital platform combining recombinase polymerase amplification (RPA) and CRISPR technology for rapid, sensitive nucleic acid quantification. The system offers a convenient "sample-to-answer" solution for point-of-care testing.
Area of Science:
- Biotechnology
- Molecular Diagnostics
- Point-of-Care Testing
Background:
- CRISPR-based nucleic acid detection offers high specificity and sensitivity but often lacks quantitative capabilities.
- Digital PCR enhances CRISPR sensitivity and quantification but faces integration challenges.
- Existing methods require complex integration for sensitive, quantitative nucleic acid analysis.
Purpose of the Study:
- To develop an integrated, one-pot digital platform for quantitative nucleic acid detection using recombinase polymerase amplification (RPA) and CRISPR technology.
- To enable sensitive and rapid "sample-to-answer" nucleic acid quantification for point-of-care applications.
- To overcome the limitations of conventional CRISPR detection methods by enhancing quantitative analysis.
Main Methods:
- Integration of nucleic acid extraction, reagent mixing, droplet generation, and isothermal reaction on a microfluidic chip.
- Development of a one-pot digital recombinase polymerase amplification (RPA)/CRISPR reaction system optimized for droplet microfluidics.
- Utilizing a smartphone-based device for temperature control and fluorescence imaging, coupled with cloud processing for real-time analysis.
Main Results:
- Demonstrated a one-pot digital RPA/CRISPR nucleic acid detection platform with "sample-to-answer" capability.
- Achieved sensitive digital quantitative detection of SARS-CoV-2 samples within 50 minutes, with a limit of detection as low as 1 copy/μL.
- Results showed comparability to commercial instruments, highlighting the platform's accuracy and reliability.
Conclusions:
- The developed integrated microfluidic platform offers a novel, highly sensitive solution for nucleic acid quantification.
- This approach significantly advances point-of-care testing (POCT) by providing rapid, quantitative, and user-friendly nucleic acid detection.
- The combination of IFAST, droplet digital RPA/CRISPR, and smartphone integration paves the way for accessible molecular diagnostics.
