提纯小管福寿螺DNA6种方法的比较研究  

Comparison on the methods of DNA extraction from lungs of Pomacea canaliculata used in PCR assay

作　　者：危芙蓉[1] 吕山[1] 张仪[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室,上海200025

出　　处：《中国人兽共患病学报》2009年第12期1181-1185,共5页Chinese Journal of Zoonoses

基　　金：“十一五”国家重大专项(2008ZX10004-010);科技部国家自然科技资源基础平台项目(2005DKA21104);“十五”国家科技攻关计划项目(2003BA71ZA09-01)联合资助

摘　　要：目的以不同方法提纯小管福寿螺DNA,用PCR检测以比较其效果。方法将80只野外采集的小管福寿螺分8组,取出肺囊并剪碎后分别用QIAGEN、天根、OMEGA的基因组DNA抽提试剂盒,及异硫酸氰胍抽提法、树脂抽提法和树脂-二氧化硅抽提法提纯小管福寿螺基因组DNA,用PCR扩增福寿螺16s rDNA,测定目的条带相对浓度以评价各方法的抽提效果。结果各种方法提纯的基因组DNA均能用PCR扩增方法扩增出目的条带,但目的条带浓度有一定差异。其中,QIAGEN公司和OMEGA公司的抽提试剂盒、树脂抽提法和树脂-二氧化硅抽提法提纯的模板PCR扩增量较高,与其余4种抽提方法比较有统计学意义。试剂盒抽提法的单价成本是手工抽提法的8.4～57.1倍。结论QIAGEN和OMEGA公司的基因组DNA抽提试剂盒的提纯效果好,但成本较高,适用于少量样本的抽提。树脂抽提法和树脂-二氧化硅抽提法从提纯效果、成本等方面评价,较适用于大量样本抽提处理工作。异硫酸氰胍抽提法,抽提快,成本低但所用试剂对身体有毒害,较适用于大量样本或应急样本的处理。To compare the efficiency of methods of DNA extraction from lungs of Pomacea canaliculata used in PCR as say, 80 P. canaliculata collected in field were divided into 8 groups and the lungs of each snail were separated from the soft body. Six methods of DNA extraction from lungs of P. canaliculata were used to extract DNA from lungs, i.e. With Qiagen, Tiangen, and Omega commercial DNA extraction kits, guanidine thiocyanate method, Chelex 100 resin method and Chelex-silica particle method. The 16S rDNA of C. canaliculata was amplified by PCR and the concentration of PCR-products relative to marker was determined in order to evaluate the efficiency of each method. It was demonstrated that each method was valid to extract DNA from lungs used in PCR assay, but the concentrations of PCR-products were different. The concentrations of PCR-products obtained by Qiangen kit, Omega kit, Chelex 100 resin method and Chelex-silica particle method were significantly higher than those of other 4 methods of DNA extraction, in which Qiangen and Omega kits were suitable for small sample size. In term of efficiency and cost, Chelex 100 method and Chelex-silica particle method were feasible for large sample scale, while the guanidine thiocyanate method was preferred due to its fast extraction and low cost, but on account of its toxicity, it is used in urgent status or in large scale of sample extraction.

关 键 词：小管福寿螺 肺囊 PCR DNA 提纯 效果 

分 类 号：R383.1[医药卫生—医学寄生虫学]