Ma Jinju, Zhang Chunyun, Liu Fuguo, Liu Yin, Wang Yuanyuan, Chen Guofu
College of Oceanology, Harbin Institute of Technology (Weihai), Wenhua West Road, 2#, Weihai, 264209, People's Republic of China.
School of Environment, Harbin Institute of Technology, Harbin, 264209, People's Republic of China.
Environ Sci Pollut Res Int. 2023 Jan;30(4):10346-10359. doi: 10.1007/s11356-022-22856-6. Epub 2022 Sep 8.
In recent years, Prorocentrum donghaiense, as a dominant species, has ranked first in terms of cumulative number and area of algal blooms in the East China Sea. In this study, the D1-D2 region of the large ribosomal subunit of P. donghaiense was used as the target gene, and specific primers DH-FP/DH-RP were designed according to the species-specific region of the target gene. An easy, sensitive and visual detection method refered to as polymerase chain reaction-nucleic acid chromatography strip (PCR-NACS) was established for P. donghaiense. The optimized parameters of the PCR amplification system are as follows: primer concentration, 0.15 μM; annealing temperature, 62 °C; and Mg concentration, 1.5 mM. The specificity test showed that PCR-NACS was exlusively specific for the detection of the target algae. The sensitivity test show that the lowest detection limit (LDL) of PCR-NACS was 2.7 × 10 ng·μL for genomic DNA and 3.58 × 10 copies·μL for plasmid DNA, respectively. The tests using both genomic DNA and plasmid DNA as templates showed that the sensitivity of PCR-NACS was 10 times higher than that of ordinary PCR. The stability test showed that the interfering algal species did not affect the detection of the target algae by PCR-NACS. In addition, the test with simulated natural samples containing target algae showed that the LDL of PCR-NACS could reach 1.27 × 10 cells·mL. In summary, the PCR-NACS established in this study may provide a new method for easy identification of P. donghaiense in natural water samples.
近年来，东海原甲藻作为优势种，在东海赤潮累计发生次数和面积方面均位居第一。本研究以东海原甲藻大核糖体亚基的D1-D2区域为靶基因，根据靶基因的种特异性区域设计了特异性引物DH-FP/DH-RP。建立了一种简便、灵敏且可视化的检测方法，即聚合酶链反应-核酸色谱条（PCR-NACS）用于检测东海原甲藻。PCR扩增体系的优化参数如下：引物浓度0.15 μM；退火温度62℃；Mg浓度1.5 mM。特异性试验表明，PCR-NACS仅对目标藻类具有特异性检测能力。灵敏度试验表明，PCR-NACS对基因组DNA的最低检测限（LDL）为2.7×10 ng·μL，对质粒DNA的最低检测限为3.58×10拷贝·μL。以基因组DNA和质粒DNA为模板的试验表明，PCR-NACS的灵敏度比普通PCR高10倍。稳定性试验表明，干扰藻类物种不影响PCR-NACS对目标藻类的检测。此外，对含有目标藻类的模拟自然样品进行的试验表明，PCR-NACS的最低检测限可达1.27×10细胞·mL。综上所述，本研究建立的PCR-NACS可为天然水样中东海原甲藻的简易鉴定提供一种新方法。
