The fish-killing alga Heterosigma akashiwo is a globally distributed, toxic, and bloom-forming raphidophyte that has caused great losses to the fishing industry in many coastal countries. Therefore, rapid and sensitive detection methods should be developed to present timely warning of harmful algal blooms. In this study, hyperbranched rolling circle amplification (HRCA) was established for the detection of H. akashiwo and compared with loop-mediated isothermal amplification (LAMP) in terms of specificity and sensitivity. The partial D1–D2 sequence of the large subunit (LSU) of rDNA of H. akashiwo was used to design a specific padlock probe for HRCA and two pairs of specific primers for LAMP. The parameters for HRCA were optimized. Cross-reactivity tests showed that the specificity of the developed HRCA for H. akashiwo was greater than that of LAMP in this study. The sensitivities of HRCA and LAMP were comparable and were 10-fold higher than that of regular PCR. These methods also yielded a detection limit of 20 fg/μL for the recombinant plasmid containing the target LSU D1–D2 and 1 cell for target species. The test with the simulated field samples indicated that the developed HRCA obtained a detection limit of 5 cells mL⁻¹, which was lower than the warning cell density (100 cells mL⁻¹) of H. akashiwo. The visual detection of positive HRCA could be achieved via coloration reaction with the addition of fluorescent SYBR Green I dye to the amplification products. The developed HRCA was also efficient for field samples with target cell densities ranging from 10 cells mL⁻¹ to 1000 cells mL⁻¹. Therefore, the proposed HRCA detection protocols are possibly applicable to the field monitoring of H. akashiwo.

杀死鱼类的藻类Heterosigma akashiwo是全球分布的，有毒的，会形成花的鳞生植物，对许多沿海国家的渔业造成了巨大损失。因此，应开发出快速灵敏的检测方法，以及时警告有害藻华。在这项研究中，建立了超支化滚环扩增（HRCA）来检测akashiwo，并将其与环介导的等温扩增（LAMP）进行了特异性和敏感性比较。赤子肠杆菌rDNA大亚基（LSU）的D1-D2部分序列用于设计针对HRCA的特异性挂锁探针和针对LAMP的两对特异性引物。优化了HRCA的参数。交叉反应测试表明，在本研究中，开发的HRCA对明石麻杆菌的特异性大于LAMP的特异性。HRCA和LAMP的敏感性相当，并且比常规PCR的敏感性高10倍。这些方法还对含有目标LSU D1-D2的重组质粒和目标物种的1个细胞的检出限为20 fg /μL。用模拟的现场样品进行的测试表明，开发的HRCA的检出限为5个细胞mL ^-1，低于赤子芥的警告细胞密度（100个细胞mL ^-1）。。通过在扩增产物中添加荧光SYBR Green I染料进行显色反应，可以目测检测到阳性HRCA。所开发的HRCA对目标细胞密度范围从10个细胞mL ^-1到1000个细胞mL ^-1的野外样品也非常有效。因此，提出的HRCA检测协议可能适用于akashiwo的现场监测。