Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology
The recombinase polymerase amplification (RPA)-CRISPR-Cas12a-FQ system enables sensitive detection of environmental DNA (eDNA) in rare fish species. Here, we present a protocol for eDNA amplification and Cas12a for target recognition using RPA. We describe steps for identifying a target site, synthesis and purification of CRISPR RNA (crRNA), and RPA isothermal amplification. We then detail procedures for constructing the eDNA CRISPR-Cas12a detection system and verifying its sensitivity. This protocol offers a high-sensitivity approach for monitoring aquatic biodiversity and conservation efforts, even in low eDNA concentrations. For complete details on the use and execution of this protocol, please refer to Wei et al.<sup>1</sup>.
Authors
- Pál Hamar (ORCID: https://orcid.org/0009-0008-8651-3013)
- Pei Yang (ORCID: https://orcid.org/0000-0001-8926-9695)
- De‐Sheng Pei (ORCID: https://orcid.org/0000-0001-6408-9575)
- Xingyi Wei (ORCID: https://orcid.org/0000-0002-1334-7359)
- Liu Li (ORCID: https://orcid.org/0000-0002-4898-3013)
Institutions
- Semmelweis University (HU)
- Chongqing 2D Materials Institute (China) (CN)
- Chongqing Public Health Medical Center (CN)
- Chongqing Medical University (CN)
Publication Details
- Journal
- STAR Protocols
- Published
- 2025-01-10
- DOI
- https://doi.org/10.1016/j.xpro.2024.103544
- Citations
- 4
- Primary Topic
- Environmental DNA in Biodiversity Studies
- Type
- article
- Field-Weighted Citation Impact
- 6.87
Funders
- Chongqing Medical University
- Centre Scientifique et Technique du Bâtiment