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A one-pot method for universal Dengue virus detection by combining RT-RPA amplification and CRISPR/Cas12a assay
Yunkai Zhang1,2, Yan Xiang3, Dengyong Hou4
1Naval Medical Center, Naval Medical University, 880 Xiangyin Road, Shanghai, 200433, China. georgezhang@smmu.edu.cn.
BMC Microbiology
|March 25, 2025
Summary
A new one-pot assay combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a rapidly detects all Dengue Virus (DENV) serotypes with high sensitivity. This simple, equipment-free method offers a promising tool for global DENV diagnostics.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Dengue Virus (DENV) poses significant global public health challenges.
- Existing diagnostic methods for DENV lack the speed, simplicity, and accuracy required for clinical practice.
Purpose of the Study:
- To develop a novel, rapid, and highly sensitive diagnostic method for universal Dengue Virus detection.
- To overcome the limitations of traditional DENV detection techniques.
Main Methods:
- Integration of recombinase polymerase amplification (RPA) assay and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas12a system into a one-pot reaction.
- Development of a universal Dengue Virus RT-RPA-CRISPR/Cas12a-lateral flow dipstick (LFD) platform.
Main Results:
- The one-pot assay achieved high sensitivity (Limit of Detection: 91.7 copies/test) and specificity for all four DENV serotypes within 40 minutes.
- The assay demonstrated no cross-reactivity with other common viruses (VSV, SeV, HSV-1, IAV).
- The LFD platform successfully identified all DENV serotypes with a sensitivity of approximately 250 copies/test.
Conclusions:
- The developed RT-RPA-CRISPR/Cas12a method provides a robust, rapid, and equipment-free alternative for universal Dengue Virus detection.
- This approach offers valuable insights for the development of diagnostic tools for other viral pathogens.
