Abstract：
BACKGROUND: Harmful algal blooms (HABs), caused by the rapid proliferation or aggregation of microorganisms, are catastrophic for the environment. The Prymnesium parvum is a haptophyte algal species that is found worldwide and is responsible for extensive blooms and death of larval amphibians and bivalves, causing serious negative impacts on the ecological environment. For the prevention and management of environmental pollution, it is crucial to explore and develop early detection strategies for HABs on-site using simple methods. The major challenge related to early detection is the accurate and sensitive detection of algae present in low abundance.
RESULTS: Herein, recombinase polymerase amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats and Cas12a protein (CRISPR-LbaCas12a) systems, and the lateral flow dipstick (LFD) was used for the first time for early detection of P. parvum. The internal transcribed spacer (ITS) of P. parvum was selected as the target sequence, and the concentration of single-strand DNA reporters, buffer liquid system, reaction time, and amount of gold particles were optimized. The RPA-CRISPR-LbaCas12a-LFD approach demonstrated highly specificity during experimental testing, with no cross-reaction against different microalgae used as controls. In addition, the lowest detection limit was 10,000 times better than the lowest detection limit of the standalone RPA approach. The feasibility and robustness of this approach were further verified by using the different environmental samples. It also observed that P. parvum are widely distributed in Chinese Sea, but the cell density of P. parvum is relatively low (<0.1 cells/mL).
CONCLUSIONS: The developed approach has an excellent specificity and offers 10,000 times better sensitivity than the standalone RPA approach. These advantages make this approach suitable for early warning detection and prevention of HAB events in environmental water. Also, the outcomes of this study could promote a shift from traditional laboratory-based detection to on-site monitoring, facilitating early warning against HABs.
RESULTS: Herein, recombinase polymerase amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats and Cas12a protein (CRISPR-LbaCas12a) systems, and the lateral flow dipstick (LFD) was used for the first time for early detection of P. parvum. The internal transcribed spacer (ITS) of P. parvum was selected as the target sequence, and the concentration of single-strand DNA reporters, buffer liquid system, reaction time, and amount of gold particles were optimized. The RPA-CRISPR-LbaCas12a-LFD approach demonstrated highly specificity during experimental testing, with no cross-reaction against different microalgae used as controls. In addition, the lowest detection limit was 10,000 times better than the lowest detection limit of the standalone RPA approach. The feasibility and robustness of this approach were further verified by using the different environmental samples. It also observed that P. parvum are widely distributed in Chinese Sea, but the cell density of P. parvum is relatively low (<0.1 cells/mL).
CONCLUSIONS: The developed approach has an excellent specificity and offers 10,000 times better sensitivity than the standalone RPA approach. These advantages make this approach suitable for early warning detection and prevention of HAB events in environmental water. Also, the outcomes of this study could promote a shift from traditional laboratory-based detection to on-site monitoring, facilitating early warning against HABs.
摘要：
背景：有害藻华（HAB），由微生物的快速增殖或聚集引起，对环境来说是灾难性的。Prymnesiumparvum是一种在世界范围内发现的藻类物种，负责幼虫两栖动物和双壳类动物的大量繁殖和死亡，对生态环境造成严重的负面影响。为了防止和管理环境污染，使用简单的方法探索和制定HABs的现场早期检测策略至关重要。与早期检测相关的主要挑战是准确和灵敏地检测低丰度的藻类。
结果：这里，重组酶聚合酶扩增（RPA）与成簇的规则间隔短回文重复序列和Cas12a蛋白（CRISPR-LbaCas12a）系统相结合，首次使用侧流试纸(LFD)对小疟原虫进行早期检测。选择细小疟原虫的内部转录间隔区（ITS）作为靶序列，以及单链DNA报告基因的浓度，缓冲液系统,反应时间,优化了金颗粒的用量。RPA-CRISPR-LbaCas12a-LFD方法在实验测试过程中表现出高度特异性，对用作对照的不同微藻没有交叉反应。此外,最低检测限比独立RPA方法的最低检测限好10,000倍.利用不同的环境样本进一步验证了该方法的可行性和鲁棒性。它还观察到细小疟原虫在中国海域广泛分布，但细小疟原虫的细胞密度相对较低(<0.1个细胞/mL)。
结论：开发的方法具有出色的特异性，并且比独立的RPA方法具有10,000倍的灵敏度。这些优点使这种方法适用于环境水中HAB事件的预警检测和预防。此外，这项研究的结果可以促进从传统的实验室检测到现场监测的转变，促进对HAB的早期预警。
结果：这里，重组酶聚合酶扩增（RPA）与成簇的规则间隔短回文重复序列和Cas12a蛋白（CRISPR-LbaCas12a）系统相结合，首次使用侧流试纸(LFD)对小疟原虫进行早期检测。选择细小疟原虫的内部转录间隔区（ITS）作为靶序列，以及单链DNA报告基因的浓度，缓冲液系统,反应时间,优化了金颗粒的用量。RPA-CRISPR-LbaCas12a-LFD方法在实验测试过程中表现出高度特异性，对用作对照的不同微藻没有交叉反应。此外,最低检测限比独立RPA方法的最低检测限好10,000倍.利用不同的环境样本进一步验证了该方法的可行性和鲁棒性。它还观察到细小疟原虫在中国海域广泛分布，但细小疟原虫的细胞密度相对较低(<0.1个细胞/mL)。
结论：开发的方法具有出色的特异性，并且比独立的RPA方法具有10,000倍的灵敏度。这些优点使这种方法适用于环境水中HAB事件的预警检测和预防。此外，这项研究的结果可以促进从传统的实验室检测到现场监测的转变，促进对HAB的早期预警。
