Revolutionizing aquatic eco-environmental monitoring: Utilizing the RPA-Cas-FQ detection platform for zooplankton

作者全名：Hu, Huan; Liu, Li; Wei, Xing-Yi; Duan, Jin-Jing; Deng, Jiao-Yun; Pei, De-Sheng

作者地址：[Hu, Huan; Wei, Xing-Yi] Chongqing Jiaotong Univ, Chongqing 400074, Peoples R China; [Hu, Huan; Wei, Xing-Yi] Univ Chinese Acad Sci, Chongqing Inst Green & Intelligent Technol, Chinese Acad Sci, Chongqing Sch, Chongqing 400714, Peoples R China; [Liu, Li] Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Key Lab Engn Biol Low Carbon Mfg, Tianjin 300308, Peoples R China; [Duan, Jin-Jing] Chongqing Miankai Biotechnol Res Inst Co Ltd, Chongqing 400025, Peoples R China; [Duan, Jin-Jing; Deng, Jiao-Yun; Pei, De-Sheng] Chongqing Med Univ, Sch Publ Hlth, Chongqing 400016, Peoples R China

通信作者：Pei, DS (通讯作者)，Chongqing Med Univ, Sch Publ Hlth, Chongqing 400016, Peoples R China.

来源：SCIENCE OF THE TOTAL ENVIRONMENT

ESI学科分类：ENVIRONMENT/ECOLOGY

WOS号：WOS:001234489100001

JCR分区：Q1

影响因子：8.2

年份：2024

卷号：929

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：RPA; CRISPR/Cas12a; Zooplankton; Species identification; eDNA; Ecological & environmental monitoring

摘要：The integration of recombinase polymerase amplification (RPA) with CRISPR/Cas technology has revolutionized molecular diagnostics and pathogen detection due to its unparalleled sensitivity and trans-cleavage ability. However, its potential in the ecological and environmental monitoring scenarios for aquatic ecosystems remains largely unexplored, particularly in accurate qualitative/quantitative detection, and its actual performance in handling complex real environmental samples. Using zooplankton as a model, we have successfully optimized the RPA-CRISPR/Cas12a fluorescence detection platform (RPA-Cas-FQ), providing several crucial "technical tips". Our findings indicate the sensitivity of CRISPR/Cas12a alone is 5 x 10(9) copies/reaction, which can be dramatically increased to 5 copies/reaction when combined with RPA. The optimized RPA-Cas-FQ enables reliable qualitative and semi-quantitative detection within 50 min, and exhibits a good linear relationship between fluorescence intensity and DNA concentration (R-2 = 0.956-0.974***). Additionally, we developed a rapid and straightforward identification procedure for single zooplankton by incorporating heat-lysis and DNAbarcode techniques. We evaluated the platform's effectiveness using real environmental DNA (eDNA) samples from the Three Gorges Reservoir, confirming its practicality. The eDNA-RPA-Cas-FQ demonstrated strong

基金机构：Chongqing Medical University Talent Project [R4014]; Chongqing Post- doctoral Innovation Mentor Studio [X7928]

基金资助正文：This study received financial support from the Chongqing Medical University Talent Project (No. R4014 to D.S.P.) , and Chongqing Post- doctoral Innovation Mentor Studio (X7928 D.S.P.) . Acknowledgments are extended to the editor and anonymous reviewers for their insightful comments, enhancing the overall quality of this work.