干血滤纸片试剂盒和水煮法提取间日疟原虫DNA用于PCR检测的比较

A comparative study of dried blood spot genomic DNA isolation kit and boiling method in DNA extraction and PCR detection of Plasmodium vivax

目的:比较水煮法和干血滤纸片基因组DNA分离试剂盒提取间日疟原虫DNA用于PCR检测及克隆研究的差异。方法:采集间日疟患者末梢血制备干血滤纸片,分别用水煮法和QIAamp DNAmini kit试剂盒提取间日疟原虫基因组DNA10份。PCR扩增LDH基因,并克隆质粒pGEM-PvLDH。分析比较2种提取方法的差异。结果:水煮法和试剂盒法提取的间日疟原虫gDNA,均扩增出LDH基因特异条带,但试剂盒提取的gDNA目的条带亮于水煮法。结论:水煮法操作简便、快速、经济,在等量血源条件下,所得DNA量较少、纯度较低;试剂盒提取可获得较高的得率,在定量检测和复合扩增时受到的影响因素较少,成功率较高。

Objective：To compare the differences of dried blood spot（DBS） genomic DNA isolation kit and the boiling method in DNA extraction,PCR and cloning of Plasmodium vivax.Methods：The peripheral blood was collected from vivax malaria patients and the dried blood spots were prepared.The genomic DNA of ten samples was extracted with boiling method and QIAamp DNA mini kit（QIAGEN,Germany）.The lactate dehydrogenase（LDH） gene was amplified by using the extracted DNA as template.The amplified fragment was purified and cloned into pGEM vector.The differences of both methods were analyzed and compared.Results：The target fragment of LDH gene was amplified with the gDNA extracted by the both methods;but the electrophoretic bands with the template by kit method were obviously brighter than that by boiling method.Conclusions：The boiling method is a simple,rapid and economical way in extracting gDNA from the dried blood spots;but in a given amount of blood sample,the extraction rate and purity of DNA are lower;while the extraction kit,with a higher extraction rate,is less affected by in the quantitative analysis or multiplex PCR.