应用qPCR方法检测中国近海塔玛亚历山大藻复合种的研究  

Application of qPCR method in detection of Alexandrium tamarense species complex in China

作　　者：高岩[1,2] 于仁成[1] 张清春[1] 周名江[1] 

机构地区：[1]中国科学院海洋研究所海洋生态与环境科学重点实验室,青岛266071 [2]中国科学院大学,北京100049

出　　处：《环境科学学报》2013年第8期2256-2263,共8页Acta Scientiae Circumstantiae

基　　金：国家自然科学基金面上项目(No.41176100);国家自然科学基金委创新研究群体基金项目(No.41121064);国家重点基础研究发展(973)计划项目(No.2010CB428705)~~

摘　　要：亚历山大藻属的部分藻种(Alexandrium spp.)能够产生麻痹性贝毒毒素(PST),是重要的有害藻华(HAB)原因种.由于亚历山大藻属中的有毒和无毒藻种形态相似,且海水中亚历山大藻的细胞密度通常很低,因此,高灵敏度、高特异性的分子生物学方法在亚历山大藻检测方面具有重要的应用前景.塔玛亚历山大藻和链状亚历山大藻是中国近海主要的有毒亚历山大藻藻种,大多属于塔玛亚历山大藻复合种第四类核糖体型(GroupⅣ,以往称为"亚洲温带核糖体型").因此,本研究尝试将实时荧光定量PCR(qPCR)方法用于我国近海塔玛亚历山大藻复合种(GroupⅣ)的快速检测,并对方法的特异性和灵敏度进行了检验.研究表明,所建立的qPCR方法能够特异性地检测我国近海不同海域分离的塔玛亚历山大藻复合种(GroupⅣ),方法具有良好的线性响应关系和较高的灵敏度,最低可检出5个藻细胞,具有良好的应用前景.然而,实验也发现,自我国近海分离的塔玛亚历山大藻复合种的不同藻株之间,以及处于不同生长阶段的藻细胞之间,qPCR检测结果存在显著差异,反映了目标藻核糖体RNA基因拷贝数的差异和变化对qPCR检测结果的影响.因此,建议在将qPCR方法用于不同海域亚历山大藻样品检测时,应采用该海域分离的藻株专门构建标准工作曲线,以减小定量分析误差.Several species in genus Alexandrium can produce paralytic shellfish toxins（PST）,and are important causative species of harmful algal blooms.Due to the similarity in morphological features between toxic and non-toxic species and the relatively low cell abundance of toxic Alexandrium cells presented in water column,molecular biological methods with high specificity and sensitivity have great potential in detecting toxic Alexandrium cells.A.tamarense and A.catenella are the two major toxic species presented in the coastal waters of China,and most of the strains identified so far were assigned to the Group IV ribotype（previously named as ＂Temperate Asian＂ ribotye） of the A.tamarense species complex.In this study,we developed a real-time quantitative PCR（qPCR） method to detect the A.tamarense species complex（Group Ⅳ） in China.The method showed high specificity and sensitivity for the detection and quantification of A.tamarense species complex（Group Ⅳ）.The detection limit was as low as 5 cells.However,significant differences in quantitative results were noticed among different isolated strains of A.tamarense species complex as well as the cells collected at different growth stages.This reflected potential interference from the difference or variation of rRNA gene copy number in Alexandrium cells.Thus,the strain of A.tamarense species complex isolated from a targeted area should be used to develop the standard calibration curve when applying the qPCR method in quantitative detection of A.tamarense species complex in the specific area.

关 键 词：亚历山大藻 有害藻华 实时荧光定量PCR 

分 类 号：X835[环境科学与工程—环境工程] X171.5