生物发光法测定天然水体细菌数量中游离ATP的去除  

ATP bioluminescence for determining total bacteria in natural water to remove free ATP

作　　者：王佳欣[1] 崔璐璐[1] 谢冰[1] 

机构地区：[1]华东师范大学生态与环境科学学院、上海市城市化生态过程与生态恢复重点实验室,上海200241

出　　处：《应用与环境生物学报》2014年第6期1091-1095,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(31370510);上海市科委项目(1352-0720600)资助~~

摘　　要：天然水体中存在浮游动植物等体细胞和游离ATP,会对细菌ATP的测定产生严重的干扰,所以探究体细胞和游离ATP的消除方法是生物发光法测定细菌总数中不可缺少的预处理步骤.本文研究了体细胞提取剂Tritonx-100的最适浓度和最适提取时间,游离ATP水解酶apyrase的最适浓度和对细菌ATP测定的影响,并将包含Tritonx-100和apyras e酶的体细胞排除体系应用于实际天然水体细菌数量的测定.结果显示,0.4%的Tritonx-100作用水样2 min能够有效提取水样中的体细胞;Apyrase酶浓度达到0.2 U时能够有效水解水样中的游离ATP;Apyrase酶的加入对细菌无水解作用,对细菌ATP的测定无明显影响;包含Tritonx-100和apyrase酶的体细胞排除体系能够裂解体细胞而不会对细菌ATP的测定造成显著影响;应用于实际天然水体的结果也表明,体细胞排除体系可为ATP生物发光法准确测定环境样品中的细菌数量创造条件.本研究表明,经Tritonx-100和apyrase酶处理后,基本能够排除体细胞和游离ATP的干扰,得到天然水体中的实际细菌总数.ATP bioluminescence is a simple, highly sensitive, and real-time monitoring assay to detect the total number of bacteria in sample. However, other forms of ATP in natural water usually interfere with determination of bacterial ATP. Therefore, how to eliminate other body cells and free ATP is an important preprocessing step in bioluminescence method. This research investigated the optimal concentration and the extraction time of body cell extraction agent （Triton-100）. Meanwhile, we also studied the optimal free ATP hydrolase （apyrase） concentration and its effect on bacterial ATP assay, which was also applied to the bacteria counts in the natural waters （body cell-free） system containing Tritonx-100 and apyrase. The result showed that using 0.4% of Tritonx-100 could effectively extract body cells in 2 minutes and 0.2 U apyrase could effectively hydrolyze body ceils in water samples. In addition, adding apyrase to samples did not hydrolyze bacteria and had no effect on the bacterial ATP measurement. The body-cell-free system with Tritonx-100 and apyrase could decempose body cells and had no significant impact on the bacterial ATP measurement. Besides, the practical application also proved that this system accurately detected the number of bacteria in environmental samples by ATP bioluminescence method. Therefore, by treating natural water with Tritonx-100 and apyrase, body ceils and free ATP can be completely eliminated, leading to actual bacteria counts in natural water.

关 键 词：ATP生物发光法 TRITONX-100 APYRASE 体细胞ATP 游离ATP 

分 类 号：S917.1[农业科学—水产科学]