As a bloom-forming dinoflagellate, Karenia mikimotoi is toxic and globally distributed. Frequent outbreaks of K. mikimotoi blooms usually lead to the mass mortality of pelagic and benthic marine life, resulting in large economic losses. Therefore, the simple, rapid, and highly effective monitoring of K. mikimotoi is crucial to minimize the damage caused by this alga. This study aimed to establish a convenient, cost-effective, and efficient molecular detection technique through loop-mediated isothermal amplification (LAMP) combined with chromatographic lateral flow dipstick (LFD) for the daily monitoring and real-time detection of K. mikimotoi. The internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences of K. mikimotoi were used to design and screen an optimal primer (KmLF3) and a detection probe (KmLF3HP). The optimized LAMP conditions were as follows: dNTP concentration, 1.2 mM; betaine concentration, 1 M; ratio of the inner primer to the outer primer, 8:1; magnesium ion concentration, 8 mM; amplification temperature, 59 °C; and amplification time, 60 min. Cross-reactivity tests confirmed the specificity of LAMP–LFD. The detection limit of LAMP–LFD for K. mikimotoi genomic DNA was 1.70 × 10⁻⁴ ng μL⁻¹, which is 100 times lower than that of conventional PCR. The detection limit of LAMP–LFD for the recombinant plasmid containing the LSU rDNA of K. mikimotoi was 6.21 × 10³ copies μL⁻¹, which was 10 times more sensitive than that of LAMP followed by agarose gel electrophoresis and SYBR Green I dye staining and 100 times lower than that of conventional PCR. The practicability of LAMP–LFD was validated by testing with simulated field-water samples, displaying a detection limit of 10⁻¹ cells mL⁻¹. In conclusion, the developed LAMP–LFD is a specific, sensitive, and accurate detection method and may be competent for the field detection of K. mikimotoi.

作为形成水华的鞭毛藻，三叶卡雷尼亚草有毒，分布全球。频繁发生的三本K. mimimotoi爆发通常会导致远洋和底栖海洋生物大量死亡，从而造成巨大的经济损失。因此，简单，快速，高效地监测K. mikimotoi至关重要，以最大程度地减少这种藻类造成的破坏。这项研究旨在通过环介导的等温扩增（LAMP）结合层析侧向量油尺（LFD）来建立方便，经济高效的分子检测技术，用于每日监测和实时检测K. mikimotoi。的内部转录间隔区（ITS）和大亚基（LSU）rDNA序列使用mikimotoi菌设计和筛选最佳引物（KmLF3）和检测探针（KmLF3HP）。优化的LAMP条件如下：dNTP浓度为1.2 mM；甜菜碱浓度，1 M；内部底漆与外部底漆的比例为8：1；镁离子浓度，8 mM；扩增温度59°C; 扩增时间为60分钟。交叉反应测试证实了LAMP–LFD的特异性。LAMP-LFD的用于检测极限K.藻的基因组DNA为1.70×10 ^-4 纳克μL ^-1，它比常规PCR的低100倍。含有mikimotoi LSU rDNA的重组质粒LAMP–LFD的检出限为6.21×10 ³拷贝μL ^-1，其灵敏度是LAMP的10倍，然后进行了琼脂糖凝胶电泳和SYBR Green I染色，是传统PCR的100倍。LAMP-LFD的实用性通过对模拟的野外水样进行测试得到了验证，显示出10 ^-1个 细胞mL ^-1的检出限。总之，已开发的LAMP-LFD是一种特异性，灵敏和准确的检测方法，可能可以胜任K. mikimotoi的现场检测。