Abstract：
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 × 109 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 (R2 = 0.956-0.974***). Additionally, we developed a rapid and straightforward identification procedure for single zooplankton by incorporating heat-lysis and DNA-barcode 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 consistency (Kappa = 0.43***) with eDNA-Metabarcoding in detecting species presence/absence in the reservoir. Furthermore, the two semi-quantitative eDNA technologies showed a strong positive correlation (R2 = 0.58-0.87***). This platform also has the potential to monitor environmental pollutants by selecting appropriate indicator species. The novel insights and methodologies presented in this study represent a significant advancement in meeting the complex needs of aquatic ecosystem protection and monitoring.
摘要：
重组酶聚合酶扩增（RPA）与CRISPR/Cas技术的整合由于其无与伦比的灵敏度和反式切割能力而彻底改变了分子诊断和病原体检测。然而,其在水生生态系统的生态和环境监测方案中的潜力仍未被开发，特别是在准确的定性/定量检测中，及其在处理复杂真实环境样品方面的实际性能。以浮游动物为模型，我们已经成功地优化了RPA-CRISPR/Cas12a荧光检测平台（RPA-Cas-FQ），提供几个关键的“技术提示”。我们的发现表明CRISPR/Cas12a单独的敏感性是5×109拷贝/反应，当与RPA组合时,其可以急剧增加至5个拷贝/反应。优化的RPA-Cas-FQ可在50分钟内实现可靠的定性和半定量检测，荧光强度与DNA浓度呈良好的线性关系(R2=0.956-0.974***)。此外,通过结合热裂解和DNA条形码技术，我们开发了一种快速，直接的单个浮游动物鉴定程序。我们使用来自三峡水库的真实环境DNA(eDNA)样本评估了该平台的有效性,确认其实用性。eDNA-RPA-Cas-FQ在检测储层中的物种存在/不存在时显示出与eDNA-Metabarcoding的强一致性(Kappa=0.43***)。此外，两种半定量eDNA定量技术显示出强正相关(R2=0.58-0.87***)。该平台还具有通过选择适当的指示物种来监测环境污染物的潜力。这项研究中提出的新颖见解和方法代表了在满足水生生态系统保护和监测的复杂需求方面的重大进展。
