Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology
Summary: 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 sit...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-03-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166724007093 |
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| author | Xing-Yi Wei Yang Pei Li Liu Péter Hamar De-Sheng Pei |
| author_facet | Xing-Yi Wei Yang Pei Li Liu Péter Hamar De-Sheng Pei |
| author_sort | Xing-Yi Wei |
| collection | DOAJ |
| description | Summary: 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.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-128232f6339548c1b606b3d4491d4346 |
| institution | Kabale University |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-128232f6339548c1b606b3d4491d43462025-01-12T05:25:46ZengElsevierSTAR Protocols2666-16672025-03-0161103544Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technologyXing-Yi Wei0Yang Pei1Li Liu2Péter Hamar3De-Sheng Pei4School of Public Health, Chongqing Medical University, Chongqing 400016, ChinaChongqing Miankai Biotechnology Research Institute Co., Ltd., Chongqing 400025, China; Chongqing No.11 Middle School, Chongqing 400061, ChinaSchool of Public Health, Chongqing Medical University, Chongqing 400016, ChinaInstitute of Translational Medicine, Semmelweis University, Tűzoltó Utca 37-47, 1094 Budapest, HungarySchool of Public Health, Chongqing Medical University, Chongqing 400016, China; Chongqing Miankai Biotechnology Research Institute Co., Ltd., Chongqing 400025, China; Corresponding author.Summary: 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.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166724007093molecular biologyCRISPRenvironmental sciences |
| spellingShingle | Xing-Yi Wei Yang Pei Li Liu Péter Hamar De-Sheng Pei Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology STAR Protocols molecular biology CRISPR environmental sciences |
| title | Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology |
| title_full | Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology |
| title_fullStr | Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology |
| title_full_unstemmed | Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology |
| title_short | Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology |
| title_sort | protocol for detecting edna in ecological rare fish using rpa crispr cas12a technology |
| topic | molecular biology CRISPR environmental sciences |
| url | http://www.sciencedirect.com/science/article/pii/S2666166724007093 |
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