Ultra-sensitive detection of ecologically rare fish from eDNA samples based on the RPA-CRISPR/Cas12a technology

Xing-Yi Wei; Li Liu; Huan Hu; Huang-Jie Jia; Ling-Kang Bu; De-Sheng Pei

doi:10.1016/j.isci.2023.107519

Ultra-sensitive detection of ecologically rare fish from eDNA samples based on the RPA-CRISPR/Cas12a technology

iScience. 2023 Aug 3;26(9):107519. doi: 10.1016/j.isci.2023.107519. eCollection 2023 Sep 15.

Authors

Xing-Yi Wei^{1

2}, Li Liu², Huan Hu^{1

2}, Huang-Jie Jia², Ling-Kang Bu², De-Sheng Pei³

Affiliations

¹ Key Laboratory of Hydraulic and Waterway Engineering of the Ministry of Education, School of River and Ocean Engineering, Chongqing Jiaotong University, Chongqing 400074, China.
² Chongqing Institute of Green and Intelligent Technology, Chongqing School of University of Chinese Academy of Sciences, Chinese Academy of Sciences, Chongqing 400714, China.
³ School of Public Health, Chongqing Medical University, Chongqing 400016, China.

Abstract

Environmental DNA (eDNA) research holds great promise for improving biodiversity science and conservation efforts by enabling worldwide species censuses in near real-time. Current eDNA methods face challenges in detecting low-abundance ecologically important species. In this study, we used isothermal recombinase polymerase amplification (RPA)-CRISPR/Cas detection to test Ctenopharyngodon idella. RPA-CRISPR-Cas12a detected 6.0 eDNA copies/μL within 35 min. Ecologically rare species were identified in the Three Gorges Reservoir Area (TGRA) using functional distinctiveness and geographical restrictiveness, with seven fish species (9%) classified as potentially ecologically rare including three species in this investigation. RPA-CRISPR/Cas12a-FQ outperformed high-throughput sequencing (HTS) and qPCR in detecting low-abundance eDNA (AUC = 0.883∗∗). A significant linear correlation (R² = 0.682∗∗) between RPA-CRISPR/Cas12a-FQ and HTS quantification suggests its potential for predicting species abundance and enhancing eDNA-based fish biodiversity monitoring. This study highlights the value of RPA-CRISPR/Cas12a-FQ as a tool for advancing eDNA research and conservation efforts.

Keywords: Biological sciences; Ecology; Molecular biology.