3种方法提取血膜疟原虫DNA的比较研究

发布时间：2018-01-12 15:18

本文关键词：3种方法提取血膜疟原虫DNA的比较研究　出处：《中国病原生物学杂志》2014年05期 　论文类型：期刊论文

【摘要】：目的对3种提取血膜疟原虫DNA的方法进行比较研究,筛选出从血膜中提取疟原虫DNA的最佳方法。方法血膜依次经过二甲苯脱油和乙醇脱色处理,然后分别采用Na2HPO4法、Chelex-100法和试剂盒法提取DNA。根据疟原虫小亚基核糖体RNA编码基因(18SSSU rRNA)保守区设计套式PCR引物,分别以3种方法提取的疟原虫DNA为模板进行套式PCR扩增,并对扩增产物进行测序分析。结果疟原虫新血膜(保存1年内)3种方法提取的DNA经PCR扩增后均呈阳性且测序成功,与镜检结果一致。Chelex-100法提取的旧血膜(保存1~5年)标本和试剂盒法提取的旧血膜和陈旧血膜(保存6~10年)的DNA经PCR扩增后均呈阳性且测序成功,与镜检结果一致。结论 3种方法均可成功提取新血膜疟原虫DNA,Chelex-100法可提取旧血膜的疟原虫DNA,试剂盒提取法适用于旧血膜和陈旧血膜的疟原虫DNA的提取。
[Abstract]:Objective to compare the three methods of extracting DNA from Plasmodium falciparum, and to select the best method for extracting DNA from the blood membrane. Methods the blood membrane was decolorized by xylene and ethanol respectively. Na2HPO4 method was used respectively. Chelex-100 and kit methods were used to extract DNA.The nested PCR primers were designed according to the conserved region of plasmodium small subunit ribosomal RNA coding gene (18SSSU rRNAs). The plasmodium DNA extracted by three methods was used as template for nested PCR amplification. The amplified products were sequenced and analyzed. Results the DNA extracted from the new blood membrane of Plasmodium plasmodium (preserved within one year) was all positive after PCR amplification and sequenced successfully. The results were consistent with the results of microscopic examination. Chelex-100 method was used to extract old blood membrane (preserved for 1 ~ 5 years) and kit method to extract old blood membrane and old blood membrane (preserved for 6 ~ 10 years). All the DNA were positive after PCR amplification and sequenced successfully. Conclusion all the three methods can be used to extract DNA from Plasmodium falciparum by Chelex-100 method. The kit extraction method is suitable for the extraction of DNA from old blood membrane and old blood membrane of Plasmodium falciparum.
【作者单位】：山东省医学科学院山东省寄生虫病防治研究所;济南大学山东省医学科学院医学与生命科学学院;济南大学山东省医学科学院医学与生命科学学院;
【基金】：中国全球基金疟疾项目实施性研究专项基金(No.201201055) 山东省自然科学基金项目(No.2012ZRC03040)
【分类号】：R382.31
【正文快照】： 疟疾是一种世界性严重危害人类健康的寄生虫病[1]。我国曾是疟疾高度流行区,间日疟(Plasmodi-um vivax,Pv)流行最为广泛,恶性疟(Plasmodiumfalciparum,Pf)次之。经过多年防治,疟疾流行范围明显减小,发病率已降至较低水平。山东省于1988年在全省范围内达到原国家卫生部颁布的基

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