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
Affiliations
Xing-Yi Wei
Key Laboratory of Hydraulic and Waterway Engineering of the Ministry of Education, School of River and Ocean Engineering, Chongqing Jiaotong University, Chongqing 400074, China; Chongqing Institute of Green and Intelligent Technology, Chongqing School of University of Chinese Academy of Sciences, Chinese Academy of Sciences, Chongqing 400714, China
Li Liu
Chongqing Institute of Green and Intelligent Technology, Chongqing School of University of Chinese Academy of Sciences, Chinese Academy of Sciences, Chongqing 400714, China
Huan Hu
Key Laboratory of Hydraulic and Waterway Engineering of the Ministry of Education, School of River and Ocean Engineering, Chongqing Jiaotong University, Chongqing 400074, China; Chongqing Institute of Green and Intelligent Technology, Chongqing School of University of Chinese Academy of Sciences, Chinese Academy of Sciences, Chongqing 400714, China
Huang-Jie Jia
Chongqing Institute of Green and Intelligent Technology, Chongqing School of University of Chinese Academy of Sciences, Chinese Academy of Sciences, Chongqing 400714, China
Ling-Kang Bu
Chongqing Institute of Green and Intelligent Technology, Chongqing School of University of Chinese Academy of Sciences, Chinese Academy of Sciences, Chongqing 400714, China
De-Sheng Pei
School of Public Health, Chongqing Medical University, Chongqing 400016, China; Corresponding author
Summary: 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.