用于单一藻细胞PCR扩增反应的样品保存方法比较  

Comparison of sample preservation methods in single-cell PCR experiment of algae

作　　者：李萍[1,2] 于仁成[1] 唐祥海[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,山东青岛266071 [2]中国科学院研究生院,北京100049

出　　处：《海洋科学》2008年第6期41-45,共5页Marine Sciences

基　　金：国家自然科学基金项目(4067072);国家863计划项目(2006AA09Z178)

摘　　要：用室内培养的塔玛亚历山大藻(Alexandrium tamarense ATHK藻株)作为实验对象,分别选择了5%福尔马林固定后常温保存、95%乙醇固定后常温保存、-20℃冷冻保存、鲁哥氏液(Lugols solution)固定后常温保存等方法,在5,15,30,60 d后对保存的样品进行单细胞PCR反应,并与新鲜样品进行比较。实验结果表明,用95%乙醇保存、-20℃冷冻保存、鲁哥氏液保存的样品在60 d后仍可扩增出目标条带,与新鲜样品没有差别,但是样品中的藻细胞形态有一些改变;而以5%福尔马林固定保存的样品只能在5 d后扩增出目标条带,但藻细胞形态比较完好。因此,对用于单细胞PCR实验的微藻样品,短期内(不多于5 d)可以采用福尔马林保存,而需要长期保存的样品,应当采用乙醇或鲁哥氏液固定,或者采用-20℃冷冻保存的方法。Recently, more and more molecular biological methods and techniques have been used in harmful-algal-bloom （HAB） studies. The single-cell PCR method, for example, is promising in genetic studies of HAB species which is hard to culture in the lab. However, little information is available on preservation of the algae samples used for single-cell PCR study. In this experiment, the cultured cells of Alexandriurn tarnarense were used to compare the different sample preservation methods, including formalin fixation （5%）, ethanol fixation （95%）, Lugol＇s solution fixation, and refrigeration at -20℃ ,to find the appropriate sample preservation method. The valid time for each sample preservation method was also tested, using samples preserved for 5 d, 15 d, 30 d and 60 d, respectively. It was found that algae samples preserved by ethanol fixation （95%）, Lugol＇s solution fixation or refrigeration at -20℃ for as long as 60 d could still be effectively used for single-cell PCR study. However, for formalin fixation （5%） method, only sample preserved for 5 d gave a positive single-cell PCR result. The morphological features of the algae samples were well preserved by formalin fixation （5%）. These results suggest that formalin fixation （5%） is a good choice for short-term （less than 5 d） preservation of algae samples, while 95% ethanol fixation, Lugol＇s solution fixation, or refrigeration at --20℃ are suitable for relatively long-term preservation of algae samples.

关 键 词：有害赤潮 单细胞PCR 保存方法 塔玛亚历山大藻(Alexandrium tamarense) 

分 类 号：X835[环境科学与工程—环境工程]