赤潮研究中圆海链藻实时荧光定量PCR检测方法的建立

Development of a real-time PCR method for rapid detection of Thalassiosira rotula

为建立快速、准确的鉴定和定量检测赤潮生物的方法,以圆海链藻为例,以其18S rDNA序列为寻找种特异性引物的靶区域,通过分析18S rDNA序列,设计出适合用于RFQ-PCR的引物与探针,并通过引物PCR验证确定其特异性,进而以圆海链藻荧光定量PCR的引物和探针(PrimerThalassiosira rotula和Taqman Thalassiosira rotula),建立了定量检测圆海链藻的实时荧光定量PCR检测方法(RFQ-PCR)。与传统的显微镜计数方法比较,两者所获结果无显著性差异,证明了本方法的可行性,从而为我国沿海水域赤潮问题的研究提供了良好的技术检测途径。

Red tide is a global marine environment problem, and it seriously endangers the aquiculture and marine ecology, so to identify the red tide species quickly and exactly, and detect the species quantitatively have been the key to forecast and prevent red tide. While the traditional micro-phytoplankton detection methods are time consuming and laborious, which can be difficult for long-term monitoring. To establish a corret and rapid identifying and quantitative detection method, in this study, red tide alga Thalassiosira rotula was taken as the object, the gene specific primers and DNA probe were designed based on the sequence of 18S rDNA isolated from T. rotula. Real-time fluorescent quantitative PCR （RFQ-PCR） method was developed for quantitative detection of T. rotula. The results of RFQ-PCR detection largely agreed with those of microscope counting method, which suggested that the RFQ-PCR could be served as a useful method for red tide algae detection.