3种方法提取血膜疟原虫DNA的比较研究

Research on three methods to extract Plasmodium DNA from blood smears

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NazHPO。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（18SSSUrRNA）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。

Objective To develop an efficient method for extracting Plasmodium DNA from blood smears by comparing three methods of DNA extraction. Methods Old or new Plasmodium blood smears were treated with xylene and then ethanol. DNA was extracted using Na2 HPO4, Chelex-100, or a DNA extraction kit. PCR primers were designed in accordance with the conserved region of the small subunit ribosomal RNA-encoding gene （18 s SSU rRNA） of Plasmodium. Nested PCR was performed using DNA extracted by three different methods as a template. PCR products were sequenced and characterized in detail. Results All three methods successfully extracted DNA from the new blood smears. The extracted DNA was amplified using nested PCR, and sequencing verified the Plasmodium to be Plasmodium vivax, Plasmodium falciparum, and Plasmodium ovale. Chelex-100 extraction successfully extracted DNA from five-year-old blood smears. The DNA kit successfully extracted DNA from all of the old blood smears. The extracted DNA was verified u- sing nested PCR, and sequencing indicated that the Plasmodium was P. vivax and P. falciparum. Conclusion All three methods can be used to extract DNA from new Plasmodium blood smears. Chelex-100 can be used to extract DNA from blood smears that are up to 5 years old, and a DNA extraction kit can be used to extract DNA from all old blood smears.