Wu Ganlin, Liu Fuguo, Chen Guofu, Wang Yuanyuan, Wang Yihan, Zhang Chunyun
School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Wenhua West Road, 2#, Weihai, 264209, Shandong Province, People's Republic of China.
Environ Sci Pollut Res Int. 2023 May;30(21):60500-60513. doi: 10.1007/s11356-023-26821-9. Epub 2023 Apr 10.
It is urgent to develop techniques that can simultaneously detect multiple microalgae, due to the diversity of harmful algal blooms (HABs)-forming algal species. The target algae species in this study are Heterosigma akashiwo, Prorocentrum donghaiense and Karenia mikimotoi. These algae are the dominant species that cause HABs in the East China Sea, and the multiple detection technique focusing on these three algae is not common. Therefore, this study established a multiplex polymerase chain reaction(mPCR) to diagnose the three algae, which is simple and low cost. First, the corresponding specific primers were designed based on the D1-D2 region of the large subunit (LSU) ribosomal DNA sequence. Then, mPCR was established and the reaction conditions were optimized. And the specificity, sensitivity, and stability of mPCR were evaluated. The result of specificity test showed that the established mPCR had good specificity for the target microalgae and did not cross-react with eighteen non-target microalgae. The sensitivity of experiment was 3.3 × 10 ng μL, and the established mPCR was not affected by the interfering microalgae. Moreover, the practicability evaluation of mPCR by using the simulated natural water samples showed that the detection limit of target microalgae was 100 cells mL, which could meet the demand for early warning of HABs. In summary, the established mPCR is characterized by strong specificity, good stability, and multiple analysis to detect H. akashiwo, P. donghaiense, and K. mikimotoi.
由于形成有害藻华（HABs）的藻类物种具有多样性，因此开发能够同时检测多种微藻的技术迫在眉睫。本研究的目标藻类物种为赤潮异弯藻、东海原甲藻和米氏凯伦藻。这些藻类是导致中国东海藻华的优势物种，针对这三种藻类的多重检测技术并不常见。因此，本研究建立了一种用于诊断这三种藻类的多重聚合酶链反应（mPCR），该方法简单且成本低廉。首先，基于大亚基（LSU）核糖体DNA序列的D1-D2区域设计了相应的特异性引物。然后，建立了mPCR并优化了反应条件。并对mPCR的特异性、灵敏度和稳定性进行了评估。特异性测试结果表明，所建立的mPCR对目标微藻具有良好的特异性，且与18种非目标微藻无交叉反应。实验的灵敏度为3.3×10 ng μL，所建立的mPCR不受干扰微藻的影响。此外，利用模拟天然水样对mPCR进行的实用性评估表明，目标微藻的检测限为100个细胞/mL，能够满足藻华早期预警的需求。综上所述，所建立的mPCR具有特异性强、稳定性好以及能够对赤潮异弯藻、东海原甲藻和米氏凯伦藻进行多重分析检测的特点。
