School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, Republic of Korea.
School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, Republic of Korea; Advanced Institutes of Convergence Technology, Suwon, Gyeonggi-do 16229, Republic of Korea.
Harmful Algae. 2017 Mar;63:23-31. doi: 10.1016/j.hal.2017.01.006. Epub 2017 Jan 27.
Red tides by the ichthyotoxic dinoflagellate Cochlodinium polykrikoides have caused large scaled mortality of fish and great loss in aquaculture industry in many countries. Detecting and quantifying the abundance of this species are the most critical step in minimizing the loss. The conventional quantitative real-time PCR (qPCR) method has been used for quantifying the abundance of this species. However, when analyzing >500 samples collected during huge C. polykrikoides red tides in South Sea of Korea in 2014, this conventional method and the previously developed specific primer and probe set for C. polykrikoides did not give reasonable abundances when compared with cell counting data. Thus improved qPCR methods and a new specific primer and probe set reflecting recent discovery of 2 new ribotypes have to be developed. A new species-specific primer and probe set for detecting all 3 ribotypes of C. polykrikoides was developed and provided in this study. Furthermore, because the standard curve between cell abundance and threshold cycle value (Ct) is critical, the efficiencies of 4 different preparation methods used to determine standard curves were comparatively evaluated. The standard curves were determined by using the following 4 different preparations: (1) extraction of DNA from a dense culture of C. polykrikoides followed by serial dilution of the extracted DNA (CDD method), (2) extraction of DNA from each of the serially diluted cultures with different concentrations of C. polykrikoides cultures (CCD method), (3) extraction of DNA from a dense field sample of C. polykrikoides collected from natural seawater and then dilution of the extracted DNA in serial (FDD method), and (4) extraction of DNA from each of the serially diluted field samples having different concentrations of C. polykrikoides (FCD method). These 4 methods yielded different results. The abundances of C. polykrikoides in the samples collected from the coastal waters of South Sea, Korea, in 2014-2015, obtained using the standard curves determined by the CCD and the FCD methods, were the most similar (0.93-1.03 times) and the second closest (1.16-1.33 times) to the actual cell abundances obtained by enumeration of cells. Thus, our results suggest that the CCD method is a more effective tool to quantify the abundance of C. polykrikoides than the conventional method, CDD, and the FDD and FCD methods.
赤潮导致鱼类大量死亡，给许多国家的水产养殖业造成了巨大损失。检测和定量该物种的丰度是将损失降到最低的最关键步骤。传统的定量实时 PCR(qPCR)方法已被用于定量该物种的丰度。然而，在分析 2014 年韩国南海发生的大规模 C.polykrikoides 赤潮期间收集的>500 个样本时，与细胞计数数据相比，这种传统方法和以前为 C.polykrikoides 开发的特定引物和探针集并没有给出合理的丰度。因此，必须开发改进的 qPCR 方法和新的反映最近发现的 2 个新核糖体类型的特定引物和探针集。本研究开发了一种用于检测 C.polykrikoides 所有 3 个核糖体类型的新型种特异性引物和探针集。此外，由于细胞丰度和阈值循环值(Ct)之间的标准曲线至关重要，因此比较评估了用于确定标准曲线的 4 种不同制备方法的效率。通过以下 4 种不同的制备方法确定标准曲线：(1)从 C.polykrikoides 密集培养物中提取 DNA，然后对提取的 DNA 进行连续稀释(CDD 方法)，(2)从不同浓度的 C.polykrikoides 连续稀释培养物中提取 DNA(CCD 方法)，(3)从从天然海水中采集的 C.polykrikoides 密集现场样本中提取 DNA，然后连续稀释提取的 DNA(FDD 方法)，以及(4)从具有不同浓度的 C.polykrikoides 的连续稀释现场样本中提取 DNA(FCD 方法)。这些方法产生了不同的结果。使用通过 CCD 和 FCD 方法确定的标准曲线，从 2014-2015 年韩国南海沿海采集的样本中获得的 C.polykrikoides 的丰度与通过细胞计数获得的实际细胞丰度最为相似(0.93-1.03 倍)和第二接近(1.16-1.33 倍)。因此，我们的结果表明，与传统方法、CDD 和 FDD 及 FCD 方法相比，CCD 方法是一种更有效的定量 C.polykrikoides 丰度的工具。
