产毒微囊藻的分子探针检测方法研究

项目名称： 产毒微囊藻的分子探针检测方法研究

项目编号： No.20807016

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 环境科学、安全科学

项目作者： 朱菁萍

作者单位： 华中科技大学

项目金额： 20万元

中文摘要： 蓝藻水华及其毒素不仅对环境造成极大的危害，还影响动物和人类的健康，甚至危及生命，是当今重要的环境问题之一。微囊藻是分布最广的一类水华蓝藻，其产生的微囊藻毒素是分布最广，对人类危害也最大的一类蓝藻毒素。研究、开发微囊藻和产毒微囊藻的监测、分析与预警系统具有十分重要的意义。本项目通过分析不同种属蓝藻的PC-IGS序列，构建系统发育树，确定序列中的保守性位点，设计、筛选了一组可用于微囊藻检测的种属特异性分子探针，建立了检测微囊藻的夹心杂交法。研究结果显示，该方法不受水样中其他生物或非生物组分的干扰，可替代传统的显微镜法；以藻毒素基因簇（mcy）为研究对象，设计、筛选了基于不同mcy基因的两组特异性探针，建立了可用于产毒微囊藻检测的夹心杂交法，检测的灵敏度为100 个细胞/ml。采用多种方法监测淡水湖泊中微囊藻和产毒微囊藻的数量变化，以验证方法的可靠性。研究显示，本项目建立的夹心杂交法灵敏度高，特异性强。与RT-PCR技术相比，该方法具有易操作，成本低等优点，可用于微囊藻和产毒微囊藻的定性定量检测，适于水体水样的常规监测。

中文关键词： 微囊藻；藻毒素；分子探针；夹心杂交

英文摘要： One of the most severe problems related to eutrophication in freshwater ecosystems is the occurrence of increasingly frequent blooms of cyanobacteria. Cyanobacteria of the genus Microcystis are among the most commonly reported bloom-forming species in lakes and reservoirs worldwide, which produce microcystins that have been implicated in several cases of animal and human intoxications. (1) Based on sequence analyses of phycocyanin intergenic spacers (PC-IGS) from Microcystis, Anabaena, Aphanizomenon and Planktothrix (Oscillatoria) strains, a genus-specific probe pair was designed, and a sandwich hybridization was established to quantitatively detect Microcystis, providing an alternative to traditional microscopic, morphology-based methods. Through BLAST and cyanobacterial culture tests, this probe pair was demonstrated to be specific for Microcystis. The biotic and abiotic components of the samples were of little disturbance to the sandwich hybridization assay. The results showed no distinct difference between the sandwich hybridization assay and microscopy. (2) Based on sequences analyses of mcy genes from Microcystis strains, two probe pairs were designed, and sandwich hybridization assays were established to quantitatively detect microcystin-producing Microcystis. The lowest detected concentration was 100 cell ml-1. The concentration of Microcystis and microcystin-producing Microcystis were detected with the established sandwich hybridization assays and microscopy in several lakes during 2010 and 2011. Compared with real-time PCR technology, the established method had such advantages as: (1) high sensitivity and specificity; (2) easiness to operate; and (3) not needing any costly equipment. The established sandwich hybridization assays could therefore be applied to quantitative analysis of Microcystis and microcystin-producing Microcystis.

英文关键词： Microcystis; microcystin; molecular probe; sandwich hybridization assay