人源细粒棘球蚴的RAPD分析体系建立  

Establishment of RAPD technique system on larval Echinococcus granulosus from Patient

作　　者：马学平[1] 李丽[1] 秦迎旭[1] 高建炜[1] 田涛[1] 

机构地区：[1]宁夏自治区疾病预防控制中心,宁夏银川750004

出　　处：《宁夏医学杂志》2010年第3期200-202,共3页Ningxia Medical Journal

基　　金：宁夏科技攻关重点项目(KGX-19-07-01);宁夏自然科学基金项目(Nz08150);宁夏卫生厅重点科研项目(2008)

摘　　要：目的建立PCR-RAPD反应体系,提升细粒棘球蚴随机扩增多态性DNA(RAPD)遗传多态性研究的稳定性,为研究细粒棘球蚴的分类学以及包虫病的分子流行病学奠定基础。方法采用改进的酚-氯仿抽提法提取细粒棘球蚴基因组DNA,用单因素筛选方法建立PCR-RAPD分析体系。结果采用改进的酚/氯仿抽提方法提取细粒棘球蚴基因组DNA质量高,适用于PCR-RAPD扩增分析;在25μL体系中,RAPD-PCR扩增反应各成分的最佳浓度为:dNTP 0.2-0.3 mmol/L、Taq酶2.5U、随机引物0.8-1.4μmol/L,模板为60ng。PCR扩增最佳条件为:95℃预变性5min;94℃变性30s、32℃退火45s、72℃延伸1min、共35个循环;最后72℃延伸10min;初步从20条引物中筛选出11条带型丰富的随机引物。结论酚-氯仿适用于提取细粒棘球蚴基因组DNA,优化的RAPD反应体系和筛选出的随机引物适合进行细粒棘球蚴RAPD遗传多态性分析。Objective To optimized an extraction condition of genomic DNA and established RAPD technique system on larval Echinococcus granulosus from Patient for improving the stability of random amplified polymorphic DNA （RAPD） genetic polymorphism. Methods The gcnomic DNA was extracted by using improvment phenol/chloroform method and established an optimal system of random amplified polymorphic DNA - PCR by using single factor screening method. Results The extracted genomic DNA was high quality, and it is suitable for PCR- RAPD analysis. The optimal PCR system for RAPD analysis was as follows： 0.2. -0.3mmol/L dNTP, 2.5U DNA template, 0.8μmol/L - 1.4μmol/L random primer, 2μL genomic DNA. The optimal PCR procedure was as follows： Temperature cycle was designed in former denaturation at 95℃ 5min, 35 cycles with denaturation 94℃ 30 s anealing 32℃ for 45s and extension at 72℃ 60s, and last estension at 72℃ 10min, 11 random primers amplified bands and were screened out in 20. Conclusion Phenol/ chloroform method is to apply to extract the genomic DNA and improve RAPD system and screen out random primer suitable for Genetic polymorphism of PCR - RAPD on larval Echinococcus granulosus from Patient.

关 键 词：人体寄生细粒棘球蚴 基因组DNA提取 PCR—RAPD体系 

分 类 号：R532.32[医药卫生—内科学]