Chattonella marina is one of the main algae that could cause harmful algal blooms. It has killed a large number of cultured fish in coastal areas of many countries, causing serious economic losses. Therefore, it is necessary to establish a method that can specifically detect C. marina at pre-bloom abundance, so that timely measures can be taken before this alga causes harm. In this study, a long probe, a short probe and a pair of amplification primers were first designed by using the internal transcribed spacer (ITS) sequence of C. marina as the target gene and using the CD74 gene of a distant species Gallus gallus as the base sequence. The double probes rolling circle amplification (dpRCA) system was then established with the designed probes and amplification primers. A novel detection protocol referred to as dpRCA-LFD by combining the dpRCA products and lateral flow dipstick (LFD) was finally established, which can make the detection results visible to the naked eyes. The reaction conditions of dpRCA were optimized and the optimal conditions were as follows: cycle number of ligation reaction, 12; ligation temperature, 58 °C; amplification temperature, 60 °C; and amplification time, 60 min. The specificity test that was performed using the optimized dpRCA conditions indicated that dpRCA-LFD was exclusively specific for the target alga. The tests with the genomic DNA of C. marina and the recombinant plasmid containing the ITS sequence of C. marina showed that the sensitivity of dpRCA-LFD was 100 times higher than that of conventional PCR. The detection limit (DL) for the genomic DNA was 8.3 × 10⁻³ ng µL⁻¹ (8.3 × 10⁻³ ng per reaction), and the DL for the recombinant plasmid DNA was 7.8 copies µL⁻¹ (7.8 copies per reaction). The practicality of the developed dpRCA-LFD was further validated by test with the spiked samples containing C. marina and field samples. The simulative test showed that the dpRCA-LFD has a DL of 10 cells mL⁻¹. The dpRCA-LFD could successfully recognize the target cells from the field samples. In summary, the dpRCA-LFD established in this study has advantages of good specificity, high sensitivity, and easily visible detection results, and therefore is promising for the analysis of C. marina in field samples.

Chattonella码头是可能导致有害藻华的主要藻类之一。它杀死了许多国家沿海地区的大量养殖鱼类，造成了严重的经济损失。因此，有必要建立一种能够在花前富集时特异性检测梭状芽胞杆菌的方法，以便在这种藻类造成危害之前及时采取措施。在这项研究中，首先使用滨海假丝酵母的内部转录间隔区（ITS）序列作为靶基因，并使用远距离种鸡种的CD74基因设计了长探针，短探针和一对扩增引物。作为基本序列。然后用设计的探针和扩增引物建立双探针滚环扩增（dpRCA）系统。通过建立dpRCA产品和侧向量油尺（LFD）的组合，最终建立了一种新的检测方法dpRCA-LFD，可以使检测结果肉眼可见。对dpRCA的反应条件进行了优化，最佳条件为：连接反应的循环数为12；结扎温度为58°C; 放大温度60°C; 扩增时间为60分钟。使用优化的dpRCA条件进行的特异性测试表明dpRCA-LFD专门针对目标藻类。用滨海梭菌的基因组DNA进行测试和含有ITS的序列的重组质粒C.码头表明dpRCA-LFD的灵敏度比常规PCR的高100倍。基因组DNA的检出限（DL）为8.3×10 ^-3  ng µL ^-1（ 每个反应8.3×10 ^-3 ng），重组质粒DNA的DL为7.8拷贝µL ^-1（每个反应7.8拷贝） ）。发达dpRCA-LFD的实用性进一步通过测试与含有加标样品验证C.码头和野外样品。模拟测试表明dpRCA-LFD的DL为10个细胞mL ^-1。dpRCA-LFD可以成功地从野外样本中识别目标细胞。综上所述，本研究建立的dpRCA-LFD具有特异性好，灵敏度高，检测结果容易可见的优点，因此有望用于野外样品中C. marina的分析。