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Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology
Xing-Yi Wei1, Yang Pei2, Li Liu1
1School of Public Health, Chongqing Medical University, Chongqing 400016, China.
STAR Protocols
|January 12, 2025
Summary
This study introduces a sensitive recombinase polymerase amplification (RPA)-CRISPR-Cas12a-FQ system for detecting environmental DNA (eDNA) from rare fish. The protocol aids in monitoring aquatic biodiversity, even with low eDNA levels.
Area of Science:
- Environmental DNA (eDNA) analysis
- Molecular biology techniques
- Biodiversity monitoring
Background:
- Detecting rare fish species using environmental DNA (eDNA) is crucial for conservation.
- Existing methods may lack the sensitivity required for low eDNA concentrations.
Purpose of the Study:
- To present a detailed protocol for a highly sensitive recombinase polymerase amplification (RPA)-CRISPR-Cas12a-FQ system.
- To enable robust detection of eDNA from rare aquatic species.
Main Methods:
- Utilized recombinase polymerase amplification (RPA) for isothermal eDNA amplification.
- Integrated CRISPR-Cas12a for specific target recognition.
- Developed a protocol for crRNA synthesis, purification, and system construction.
Main Results:
- Demonstrated high sensitivity in detecting eDNA using the RPA-CRISPR-Cas12a-FQ system.
- Verified the system's effectiveness in identifying target DNA sequences.
Conclusions:
- The RPA-CRISPR-Cas12a-FQ protocol offers a sensitive method for eDNA detection.
- This approach supports aquatic biodiversity monitoring and conservation efforts, especially in challenging low-concentration scenarios.
