单条线虫DNA提取方法优化-给专家电子版-审回稿修改.docVIP

单条线虫DNA提取方法 王江岭1 张建成2 顾建锋1* （1宁波出入境检验检疫局技术中心 宁波 315012 2 嘉兴出入境检验检疫局 嘉兴 314000） Method of extract DNA from a single nematode. Wang Jiangling1, Zhang Jiancheng2, Gu Jianfeng1*（Ningbo Entry-exit Inspection and Quarantine Bureau, Ningbo 315012; 2Jiaxing Entry-exit Inspection and Quarantine Bureau, Jiaxing 314000） Abstract There are many problems in extracting DNA from large number of nematodes. The nematodes could be a mixed specie.extracted DNA were decreasing in experiment process. Amplification might be cumbered by adding more solutes, and even to effect on the next experiments. In this method a single nematode was freezed by liquid nitrogen and heated at 85℃ to disrupte the structure of nematode cells by changing the temperature suddenly. Then the proteins were dissolved easily by proteinase K and more DNA were extracted. This method was proved effective, rapid, and stabile by kinds of nematodes. The advantages of this method are: (1) PCR Buffer attached to Taq enzyme take the place of WLB and SDS lysis liquids, which reduces the instability of reaction system and the effect of added solutes. (2) All the process is finished in one PCR tube, no liquid transported and less solutes added. So the pollution and bad effect to the later PCR process are greatly reduced. (3) Manual disruption of nematode is replaced by poikilothermic treatment; pollution is avoided and no experiment technique is required. Compared with two other proteinase K methods of nematode DNA extraction, this method was proved effective and stabile, and could be used in rapid test; nematode proteins were dissolved completely and more DNA released, the PCR result is good even when the extracted DNA were diluted for 32 time, which procides more molecular experiments chances. Key words nematode, optimized method, DNA, proteinase K, temperature 摘 要:用液氮快速冷冻线虫，继以85℃快速变温，用温度的剧烈变化使线虫解，再以蛋白酶K降解蛋白质，提高DNA的纯度和数量。另外两种提取线虫DNA的蛋白酶K方法，提取线虫DNA的时间短，效率高，PCR扩增结果稳定可靠，符合快速检测的需求关键词: 线虫 方法DNA 蛋白酶K由于线虫体形较小，形态学鉴定十分困难，分子