Page 48 - 《渔业研究》2025年第3期

第 3 期 吴隐生等： 基于 RAA-LbCas12a 技术的有毒（害）赤潮快速检测产品应用效果评估 305

Application evaluation of a rapid detection product for toxic and harmful red
tide based on RAA-LbCas12a

2
3
2
2
1
WU Yinsheng ，PAN Feifei ，LI Cong ，CHEN Xiaoyao ，CHEN Jianming ，WANG Lu 3*
(1. School of Environmental and Safety Engineering, Fuzhou University, Fuzhou 350108, China;
2. Fishery Resources Monitoring Center of Fujian Province, Fuzhou 350003, China;
3. Fujian Key Laboratory on Conservation and Sustainable Utilization of Marine Biodiversity, Minjiang University,
Fuzhou 350108, China)
Abstract: [Objective] In light of escalating anthropogenic activities impacting aquatic ecosystems and global
climate change, the frequency and magnitude of red tide occurrences have been steadily increasing year after
year. Red tide has evolved into a marine ecological catastrophe that poses a grave threat to oceanic ecological
environments, socio-economic development, and public health. Fujian Province possesses abundant marine reso-
urces; however, the rapid progress of marine aquaculture is hindered by the occurrence of red tide. Therefore, the
development of precise, rapid, and specific detection techniques for harmful algae is imperative to enable early
monitoring and warning systems for red tides. [Methods] The study evaluated a rapid detection method that
combined recombinase aided amplification (RAA) with LbCas12a nuclease. This method was tested using field
red tide samples and pilot applications, with Karenia mikimotoi, Karlodinium veneficum, and K. brevis serving as
representative species. The core principle of the RAA-LbCas12a method involved designing amplification
primers and crRNA that specifically targets the internal transcribed spacer (ITS) region unique to the desired
algal species, which enabled rapid amplification of the target nucleic acid, followed by analysis of the amplifica-
tion products. Additionally, lateral flow strips were utilized for visual interpretation of the detection results.
[Results] The RAA-LbCaas12a assay demonstrated high accuracy in detecting target microalgae in field red
tide samples, even at concentrations as low as 10 cells/mL (representing only 0.1% of the total cells present in
red tide samples). While the target microalgae in environmental samples were consistently detected multiple
times during the 65-day pilot application, it demonstrated a large level of concordance with the results obtained
from colloidal gold immunoassay strips and microscopic observations. Importantly, no significant instances of
false positives were observed. [Conclusion] Field application data demonstrate that the RAA-LbCas12a tech-
nology exhibits remarkable sensitivity, specificity, reliability, and operational simplicity. Additionally, this tech-
nology has proven to be highly applicable for the field detection of various red tide algae species. The RAA-Lb-
Cas12a technology only requires a portable rapid detection toolkit, enabling field inspectors to promptly per-
form on-site microalgae testing, which holds immense significance in enhancing red tide monitoring and early
warning capabilities, as well as strengthening measures for preventing and controlling marine ecological disasters.
Key words: red tide; recombinase aided amplification (RAA); LbCas12a nuclease; rapid detection