湖北钉螺基因组5种抽提方法的比较  

Comparison of five different methods of extracting genomic DNA from Oncomelania hupensis

作　　者：马琳[1,2] 李石柱[1] 杨频[1] 尤平[2] 周晓农[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,上海200025 [2]陕西师范大学生命科学学院

出　　处：《中国病原生物学杂志》2011年第2期129-132,141,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30590373);科技部重大支撑专项(No.2003DIA6N009;No.2005DKA21104;No.2007BAC03A02);国家传染病重大专项(No.2008ZX10004-011)

摘　　要：目的 5种方法提取湖北钉螺基因组DNA,PCR扩增线粒体基因组的COⅡ基因以比较其扩增效果,从而选择适合湖北钉螺大样本基因组抽提的最佳方法。方法将50只钉螺分为5组,每组10只,分别用OMEGA基因组DNA抽提试剂盒、天根海洋动物组织基因组DNA提取试剂盒、简化基因组抽提方法、CTAB法和饱和酚抽提法抽提单只湖北钉螺基因组DNA,从消化、抽提时间、抽提浓度、成本等方面评价各种方法的优劣;PCR扩增线粒体基因组COⅡ基因,测定目的条带相对浓度以衡量扩增效果。结果 5种方法均能提取基因组DNA,消化时间最短的为饱和酚法,其余均为3 h;抽提时间最短的为两种试剂盒和CTAB,最长的为简化法;抽提成本最高的为OMEGA试剂盒法,最低的为CTAB法。5种方法PCR均扩增出COⅡ基因,但目的条带浓度有一定差异,其中OMEGA公司和天根公司试剂盒及实验室简化法提纯的模板PCR扩增浓度较高。结论用OMEGA公司生产的基因组DNA抽提试剂盒抽提湖北钉螺基因组DNA效果好,但成本较高,适用于对小样本的抽提;简化法从提纯效果、成本等方面评价,较适用于大样本抽提。Objective To use 5 methods to extract genomic DNA fromOncomelania hupensisin order to determine the best method of extracting DNA from bulk samples and to compare their effectiveness using mt-COⅡ amplification.Methods Fifty specimens ofOncomelania hupensiswere divided into 5 groups that were subjected to DNA extraction with commercial OMEGA and TianGen kits,simple DNA extraction,DNA extraction with CTAB,and DNA extraction with phenol-chloroform.The extraction time,the extraction concentration,and the cost of the methods were compared.COⅡ was amplified by PCR and the concentration of PCR products relative to markers was determined in order to evaluate the effectiveness of each method.Results Each method extracted genomic DNA.DNA extraction with phenol-chloroform had the fastest digestion while DNA extraction with the OMEGA and TianGen kits and with CTAB took 3 h.DNA extraction with the OMEGA kit was the most expensive method of extraction.The concentration of PCR products differed but each method was effective at extracting DNA for PCR.DNA extraction with the OMEGA and TianGen kits and simple DNA extraction resulted in higher concentrations of PCR products.Conclusion DNA extraction with the OMEGA and TianGen kits is suitable for a small number of samples.In terms of efficiency and cost,simple DNA extraction is feasible on a large scale.

关 键 词：湖北钉螺 基因组DNA 抽提方法 

分 类 号：R38[医药卫生—医学寄生虫学]