College of Oceanology, Harbin Institute of Technology (Weihai), Wenhua West Road, 2#, Weihai, Shandong Province 264209, PR China.
College of Oceanology, Harbin Institute of Technology (Weihai), Wenhua West Road, 2#, Weihai, Shandong Province 264209, PR China; School of Marine Sciences, Ningbo University, Ningbo, 315211, PR China.
Harmful Algae. 2020 Jul;97:101857. doi: 10.1016/j.hal.2020.101857. Epub 2020 Jun 24.
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 ng µL (8.3 × 10 ng per reaction), and the DL for the recombinant plasmid DNA was 7.8 copies µL (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. 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.
海洋拟菱形藻是引发赤潮的主要藻种之一，它曾在多个国家的沿海地区大量养殖鱼类，导致严重的经济损失。因此，建立一种能够在赤潮发生前特异性检测到海洋拟菱形藻的方法非常重要，以便在该藻类造成危害之前采取及时的措施。本研究以海洋拟菱形藻的内转录间隔区（ITS）序列为靶基因，以远缘物种鸡的 CD74 基因为基础序列，设计了长探针、短探针和一对扩增引物，建立了基于设计探针和扩增引物的双探针滚环扩增（dpRCA）系统。最终，将 dpRCA 产物与侧向流试纸（LFD）相结合，建立了一种新的检测方法，称为 dpRCA-LFD，该方法可使检测结果肉眼可见。优化了 dpRCA 的反应条件，最佳反应条件为：连接反应循环数 12；连接温度 58℃；扩增温度 60℃；扩增时间 60min。优化后的 dpRCA 条件下的特异性试验表明，dpRCA-LFD 仅对目标藻类具有特异性。使用海洋拟菱形藻基因组 DNA 和含有海洋拟菱形藻 ITS 序列的重组质粒进行的试验表明，dpRCA-LFD 的灵敏度比常规 PCR 高 100 倍。dpRCA-LFD 对基因组 DNA 的检测限（DL）为 8.3×10ngµL（每个反应 8.3×10ng），对重组质粒 DNA 的 DL 为 7.8 拷贝µL（每个反应 7.8 拷贝）。用含海洋拟菱形藻的加标样品和现场样品进一步验证了所建立的 dpRCA-LFD 的实用性。模拟试验表明，dpRCA-LFD 的 DL 为 10 个细胞 mL。dpRCA-LFD 可以成功识别来自现场样品的目标细胞。总之，本研究建立的 dpRCA-LFD 具有特异性好、灵敏度高、检测结果肉眼可见等优点，有望用于现场样品中海洋拟菱形藻的分析。
