土鳖虫DNA的RAMP-PCR反应体系的建立及优化  

Establishment and optimization of RAMP-PCR reaction system for DNA of Eupolyphaga sinensis

作　　者：王慧[1] 苏双良[1] 任慧君[2] 赵志壮[1] 白秀娟[1] 

机构地区：[1]东北农业大学动物科学技术学院,黑龙江哈尔滨150030 [2]东北农业大学生命科学学院,黑龙江哈尔滨150030

出　　处：《中草药》2014年第6期835-839,共5页Chinese Traditional and Herbal Drugs

摘　　要：目的建立并优化土鳖虫基因组DNA的RAMP-PCR反应体系及扩增程序,为土鳖虫遗传多样性的研究提供依据。方法以雌性土鳖虫为材料,常规酚/氯仿提取基因组DNA。使用RAMP引物(ISSR807+RAPD A5),采用正交优化方法对影响PCR反应的Mg2+、dNTPs、引物、DNA聚合酶、模板进行体系优化,同时选择最佳的引物退火温度。结果最适引物(ISSR807+RAPD A5)组合进行土鳖虫的RAMP-PCR分析的反应体系为总体积25μL,包括10×缓冲液2.5μL、Mg2+1.00 mmol/L、dNTPs 2.00mmol/L、引物0.50μmol/L、TaqDNA聚合酶1.00 U、DNA模板1.50 ng/μL,最佳退火温度为46.8℃。结论本实验建立的最佳RAMP-PCR反应体系稳定、重复性好,可用于土鳖虫种质资源鉴定和遗传多样性研究。Objective To provide the basis for the genetic diversity research ofEupolyphaga sinensis by establishing and optimizing the random amplified microsatellite polymorphism RAMP-PCR reaction system and amplification procedure for genomic DNA. Methods Phenol chloroform extraction of genomic DNA was performed on female E. sinensis. Based on RAMP Primer （ISSR807 ＋ RAPD As）, the orthogonal test was adopted to optimize the RAMP-PCR amplification system on E. sinensis in five factors （Mg2＋, dNTPs, primer, Taq DNA polymerase, and template DNA） at four levels, and the suitable anneal temperature of the primer was selected. Results The optimal Primer （ISSRS07＋RAPD As） for RAMP-PCR system （25 μL reaction volume） of E. sinensis was： 10 × PCR buffer （2.5 μL）, Mg2＋ （1.0 mmol/L）, dNTPs （2.0 mmol/L）, primer （0.5 μmol/L）, Taq DNA polymerase （1 U）, and template DNA （1.5 ng/μL）; The optimized anneal temperature was 46.8 ℃. Conclusion The established and optimized RAMP-PCR reaction system is stable and repeatable, which could provide the basis for the analysis of germplasm resources and genetic diversity ofE. sinensis.

关 键 词：土鳖虫 正交试验 体系优化 RAMP-PCR体系 遗传多样性 

分 类 号：R282.12[医药卫生—中药学]