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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.
Abstract:
Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection has advanced rapidly due to its simplicity, high specificity, and high sensitivity. However, most conventional CRISPR-based detection methods lack quantitative capability. Although the strategy of digital PCR technology enhances the sensitivity of CRISPR detection and enables quantification analysis through sample dispersion, integration remains limited. In this study, we developed a one-pot digital recombinase polymerase amplification (RPA)/CRISPR nucleic acid detection platform by integrating RPA-assisted CRISPR nucleic acid detection with droplet microfluidics. A microfluidic chip was designed to integrate nucleic acid extraction, reagent mixing, droplet generation and collection, and on-chip isothermal reaction. The one-pot RPA/CRISPR reaction system and droplet preparation conditions were systematically optimized. In addition, a portable smartphone-based temperature control and fluorescence imaging device was developed to enable convenient "sample-to-answer" nucleic acid quantification with high sensitivity. This platform allows digital quantitative detection of SARS-CoV-2 samples within 50 min, achieving a detection limit as low as 1 copy/μL, with results comparable to commercial instruments. By integrating the immiscible filtration assisted by surface tension (IFAST)-based nucleic acid extraction with droplet digital RPA/CRISPR and utilizing smartphones and cloud processing for real-time result analysis, this study offers a novel "sample-to-answer" and highly sensitive nucleic acid quantification solution for point-of-care testing (POCT).
