School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Harbin Institute of Technology (Weihai), Wenhua West Road, 2#,, Weihai, 264209, Shandong Province, People's Republic of China.
School of Environment, Harbin Institute of Technology, Harbin, 264209, People's Republic of China.
Environ Sci Pollut Res Int. 2021 Aug;28(31):42570-42582. doi: 10.1007/s11356-021-13673-4. Epub 2021 Apr 4.
Harmful algal blooms caused by Karlodinium veneficum recently occurred with high incidence, posing a serious threat to the marine ecological environment, public health, and mariculture. It is therefore rather vital to establish a method for rapid detection of K. veneficum. In this study, the D1-D2 region of the large subunit rDNA (LSU rDNA D1-D2) of K. veneficum was cloned and sequenced to design the specific probes and primers. A novel method referred to as double-nick rolling circle amplification (dn-RCA) based on the designed probes and primers was initially established. The optimal reaction conditions for dn-RCA were as follows: probe concentration, 200 pM; ligation temperature, 57 °C; ligation time, 50 min; amplification temperature, 60 °C; and amplification time, 60 min. Furthermore, lateral flow dipstick (LFD) was employed instead of agarose gel electrophoresis to analyze dn-RCA products, which can simplify the detection procedure and reduce the operation time. The sensitivity of dn-RCA-LFD was tested with the genomic DNA, the recombinant plasmid containing the inserted LSU rDNA D1-D2, and the DNA crude extract of K. veneficum. The results showed that the sensitivity of dn-RCA-LFD was 10 times higher than that of conventional PCR; the detection limit of dn-RCA-LFD was 1.1 × 10 ng μL for the genomic DNA, 360 copies μL for the recombinant plasmid, and 5.3 cells mL for DNA crude extract. The results of the cross-reactivity test with 22 control microalgal species showed that the dn-RCA-LFD had high specificity for K. veneficum. The stability of dn-RCA-LFD was tested by mixing the interfering genomic DNA with the target genomic DNA, which can be expected to simulate the natural samples containing different ratios of interfering cells to target cells. The results indicated that the performance of dn-RCA-LFD was immune to the DNA concentration of the interfering species. Finally, the practicability of dn-RCA-LFD was further confirmed by the test with field samples collected from the East China Sea. In conclusion, the established dn-RCA-LFD has advantages of high sensitivity, strong specificity, and stable performance, and is therefore promising for rapid detection of K. veneficum.
卡盾藻属（Karlodinium veneficum）引发的有害藻华近年来频繁暴发，对海洋生态环境、公众健康和海水养殖业构成了严重威胁。因此，建立一种快速检测卡盾藻属的方法非常重要。本研究克隆并测序了卡盾藻属的大亚基 rDNA（LSU rDNA D1-D2）的 D1-D2 区，设计了特异性探针和引物。基于设计的探针和引物，我们首次建立了一种新的方法，称为双缺口滚环扩增（dn-RCA）。dn-RCA 的最佳反应条件为：探针浓度 200 pM，连接温度 57°C，连接时间 50 min，扩增温度 60°C，扩增时间 60 min。此外，我们采用侧向流试纸条（LFD）代替琼脂糖凝胶电泳来分析 dn-RCA 产物，从而简化了检测步骤，减少了操作时间。使用基因组 DNA、含插入 LSU rDNA D1-D2 的重组质粒和卡盾藻属的 DNA 粗提物对 dn-RCA-LFD 的灵敏度进行了测试。结果表明，dn-RCA-LFD 的灵敏度比常规 PCR 高 10 倍；dn-RCA-LFD 的检测限为基因组 DNA 为 1.1×10 ng μL，重组质粒为 360 拷贝 μL，DNA 粗提物为 5.3 个细胞 mL。用 22 种对照微藻进行交叉反应性测试的结果表明，dn-RCA-LFD 对卡盾藻属具有高度特异性。通过将干扰基因组 DNA 与靶基因组 DNA 混合来测试 dn-RCA-LFD 的稳定性，这可以模拟含有不同比例干扰细胞和靶细胞的天然样本。结果表明，dn-RCA-LFD 的性能不受干扰物种 DNA 浓度的影响。最后，通过对东海采集的现场样本进行测试，进一步验证了 dn-RCA-LFD 的实用性。综上所述，建立的 dn-RCA-LFD 具有灵敏度高、特异性强、性能稳定的优点，有望用于卡盾藻属的快速检测。
