建立几种赤潮藻的PCR–膜反向斑点杂交检测技术-遗传学专业论文.docxVIP

哈 哈尔滨工业大学理学硕士学位论文 II II Abstract The identification and detection of harmful algae is the premise and foundation for red tide research and warning, so its methodology becomes a hot point. However, the traditional identification method (light microscopy) based on morphological characteristics of red tide algae used to distinguishing causative species has disvantages of low efficiency and accuracy, and especially not fit for parallel detection of several algae in the natural water samples. Therefore, this study aims to develop a molecular technique—PCR-based reverse dot blot hybridization for simultaneous detection of several harmful algal species. Six common red tide algae, including Prorocentrum donghaiense Lu, Skeletonema costatum (Greville) Cleve, Symbiodinium sp., Chaetoceros debilis, Heterosigma akashiwo and Nitzschia closterium, were used as test algae in this paper. Firstly, the genomic DNA from six algae were isolated to used as template to PCR-amplifythe large subunit (LSU) D1–D2 of rDNA sequences, and then the PCR products were purified was and ligated with T-vector. The ligation product was used to transformedthe competent Escherich coli. Through blue–white spot screening, the positive colonies were used for commercial sequences. According to the obtained sequences, we designed the universal primers using Primer Premier 5.0 and the suitable probes with Array Designer 4.20. Then the probes we evaluated with Oligo, and three probes with good specificity were screened. The selected probes were further tested using reverse dot blot hybridization, and finally got the specific probes. Then the probes were labelled with digoxigenin. Six common red-tide algae and some microalgae were choosen. The specificity of the probes were confirmed by dot blot hybridization assay. The probes were tailed and were used to prepare low-density membrane-based chip. This study preliminarily established a PCR-reverse dot blot hybridization assay, which was based on the large subunit