Wang Lu, Chen Xiaoyao, Pan Feifei, Yao Guangshan, Chen Jianming
Fujian Key Laboratory on Conservation and Sustainable Utilization of Marine Biodiversity, Fuzhou Institute of Oceanography, Minjiang University, Fuzhou, China.
Fishery Resources Monitoring Center of Fujian Province, Fuzhou, China.
Front Microbiol. 2023 Aug 7;14:1205765. doi: 10.3389/fmicb.2023.1205765. eCollection 2023.
Harmful algal blooms (HABs), mainly formed by dinoflagellates, have detrimental effects on marine ecosystems and public health. Therefore, detecting HABs is crucial for early warning and prevention of HABs as well as the mitigation of their adverse effects. Although various methods, such as light microscopy, electron microscopy, real-time PCR, and microarrays, have already been established for the detection of HABs, they are still cumbersome to be exploited in the field. Therefore, rapid nucleic detection methods such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)-lateral flow dipstick (LFD) have been developed for monitoring bloom-forming algae. However, the CRISPR/Cas-based detection of HABs has yet to be applied to this field. In this study, we developed a method for detecting (), a typical ichthyotoxic dinoflagellate responsible for global blooms. Our method utilized Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) to target and cleave the internal transcribed spacer (ITS) of , guided by RNA. We leveraged the target-activated non-specific single-stranded deoxyribonuclease cleavage activity of LbCas12a to generate signals that can be detected using fluorescence-read machines or LFDs. By combining RPA and LbCas12a with reporters, we significantly enhanced the sensitivity, enabling the detection of ITS-harboring plasmids at concentrations as low as 9.8 aM and genomic DNA of at levels as low as 3.6 × 10 ng/μl. Moreover, we simplified the genomic DNA extraction method using cellulose filter paper (CFP) by directly eluting the DNA into RPA reactions, reducing the extraction time to < 30 s. The entire process, from genomic DNA extraction to result reporting, takes less than an hour, enabling the identification of nearly a single cell. In conclusion, our method provided an easy, specific, and sensitive approach for detecting , offering the potential for efficient monitoring and management of blooms.
有害藻华（HABs）主要由甲藻形成，对海洋生态系统和公众健康具有有害影响。因此，检测有害藻华对于早期预警和预防有害藻华以及减轻其不利影响至关重要。尽管已经建立了各种方法，如光学显微镜、电子显微镜、实时聚合酶链反应（PCR）和微阵列来检测有害藻华，但在现场应用它们仍然很麻烦。因此，已经开发了诸如重组酶聚合酶扩增（RPA）和环介导等温扩增（LAMP）-侧向流动试纸条（LFD）等快速核酸检测方法来监测形成藻华的藻类。然而，基于CRISPR/Cas的有害藻华检测尚未应用于该领域。在本研究中，我们开发了一种检测（）的方法，（）是一种导致全球藻华的典型鱼毒性甲藻。我们的方法利用来自毛螺菌科细菌ND2006的Cas12a（LbCas12a）在RNA引导下靶向并切割（）的内转录间隔区（ITS）。我们利用LbCas12a的靶标激活非特异性单链脱氧核糖核酸酶切割活性来产生可使用荧光读取机器或LFD检测的信号。通过将RPA和LbCas12a与报告分子相结合，我们显著提高了灵敏度，能够检测低至9.8 aM浓度的携带ITS的质粒和低至3.6×10 ng/μl水平的（）基因组DNA。此外，我们通过将DNA直接洗脱到RPA反应中简化了使用纤维素滤纸（CFP）的基因组DNA提取方法，将提取时间缩短至<30秒。从基因组DNA提取到结果报告的整个过程耗时不到一小时，能够鉴定几乎单个细胞。总之，我们的方法为检测（）提供了一种简便、特异且灵敏的方法，为有效监测和管理（）藻华提供了潜力。
