多房棘球绦虫基因组DNA三种提取方法的比较

Comparison of three methods forextracting genomic DNA from Echinococcus multilocularis

目的综合比较95℃水浴法、碱裂解法及离心柱法提取多房棘球绦虫虫体基因组DNA的质量差异,筛选出适用于PCR鉴定多房棘球绦虫线粒体DNA分型的DNA提取方法。方法无菌条件下取适量多房棘球绦虫原头节(Protoscoleces,PSCs)、囊壁组织样本为实验材料,分别采用95℃水浴法、碱裂解法及离心柱法提取虫体样本中的基因组DNA,检测其浓度与A260/A280比值,并以提取的DNA为模板,通过PCR扩增多房棘球绦虫核基因延伸蛋白样蛋白(Ezrin/radixin/moesin-like protein,elp)和线粒体基因细胞色素b(Cytochrome b,cob)、NADH脱氢酶亚基2(NADH dehydrogenase subunit 2,nad2)和细胞色素c氧化酶亚基I(Cytochrome c oxidase subunit 1,cox1)的片段,综合比较3种方法提取的基因组DNA扩增效果。结果3种方法均可从PSCs中提取出基因组DNA,琼脂糖凝胶电泳和核酸定量结果显示离心柱法提取的基因组DNA A260/A280比值最高,差异具有统计学意义(P<0.01),且在加样模板总量一致的情况下,以离心柱法提取的PSCs DNA为模板扩增的cob、nad2和cox1基因产物电泳条带清晰、明亮,片段长度准确;而使用95℃水浴法和碱裂解法提取的PSCs基因组DNA仅有效扩增出cob基因条带。通过离心柱法提取的囊壁组织DNA为模板仅扩增出elp基因片段,小鼠组织基因组DNA未扩增出任何虫体的目的基因片段。结论使用离心柱法从多房棘球绦虫PSCs中提取的基因组DNA质量最佳,能够有效扩增出虫体线粒体基因片段,且无宿主来源细胞的污染,可满足后续实验室对多房棘球绦虫不同虫株的基因型鉴定研究。

Objective The quality of DNA extracted by 95℃water bath method,alkali lysis method and centrifugal column method was compared,and the DNA extraction method suitablefor PCR identification of mitochondrial DNA typing of Echinococcus multilocularis mitochondrial DNA was selected.Methods Under aseptic conditions,the genomic DNA of Echinococcus multilocularis were extracted from protoscoleces(PSCs),cyst wall tissue samples and mouse tissue samples by 95℃water bath method,alkaline lysis method and centrifugal column method,and its concentration and the ratio of A260/A280 were detected.The extracted DNA was used as a template for PCR amplification of nuclear DNA ezrin/radixin/moesin-like protein(elp)and three mitochondrial genes(cob,nad2 and cox1)of Echinococcus multilocularis,and the extraction effects of these three methods were compared.Results Genomic DNA can be extracted from PSCs by all the above three methods,and the results of agarose gel electrophoresis and nucleic acid quantification showed that the ratio of A260/A280 of genomic DNA extracted by centrifugal column method was the significantly highest(P<0.01),and under the condition that the total amount of added templates were consistent,the electrophoretic bands of the three mitochondrial gene products(cob,nad2 and cox1)amplified by PSCs DNA extracted by centrifugal column method were clear,bright and accurate in length,while only cob gene was amplified by 95℃water bath and alkaline lysis method.However,the nuclear gene elp fragment was amplified by the DNA extracted by centrifugal column method from cyst wall.The target gene of worms fragments were not amplified by the DNA extracted by centrifugal column method from mouse tissues.Conclusion Extraction of genomic DNA from PSCs of Echinococcus multilocularis by centrifugal column method is the best method,and it could effectively amplify mitochondrial gene fragments of the worms without contamination from host-derived cells,which can meet the requirements of PCR identification of different Echinococcus multilocularis strains in laboratories.