禽波氏杆菌RAPD-PCR反应体系的建立与优化  

Establishment and Optimization of RAPD Reaction System for Bordetella avium

作　　者：杨萍萍[1] 赵雪[1] 刘冠华[1] 贺晓华[1] 朱瑞良[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国兽医杂志》2013年第11期16-19,共4页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(31272595;30972183;30740077)

摘　　要：随机扩增多态性DNA(RAPD)作为一种分子标记技术,可用于多种病原菌的基因分型。为建立适合禽波氏杆菌RAPD分析的扩增体系,本试验以禽波氏杆菌基因组DNA为模板,对20条随机引物进行了筛选,并对RAPD-PCR反应体系中的重要参数设置梯度进行单因素试验分析,以确定最佳反应条件。经大量重复试验筛选到6条随机引物,确定25μL的反应体系中模板DNA的量为50ng,随机引物浓度为0.2μmol/L,dNTPs的量为0.2mmol/L,Mg^(2+)浓度为1 mmol/L,Taq酶为1U。该优化体系对22株禽波氏杆菌均能扩增出清晰稳定且多态性好的DNA指纹图谱,为后续的禽波氏杆菌RAPD基因分型奠定了良好的基础。As a molecular marker technique, random amplified polymorphie DNA （RAPD） can be used for the genotyping of a variety of pathogens. To establish a suitable RAPD reaction system for Bordetella avium, genomic DNAs of Bordetella avium were ex- tracted as templates and 20 random primers were screened. Several significant parameters of RAPD-PCR reaction system were stud- ied and optimized by setting gradient for single-factor analysis. Through a large number of repeated trials, 6 suitable random primers were found and a stable reaction system was established： 25 IxL reaction system containing 50 ng DNA template, 0.2 p.mol/L random primer, 0.2 mmol/L dNTPs, lmmol/L Mg^2＋ and 1U Taq DNA polymerase. It can generate clear, steady and polymorphie DNA frag- ment patterns for 22 Bordetella avium isolates which laid a good foundation for the subsequent RAPD genotyping.

关 键 词：禽波氏杆菌 RAPD 反应体系 优化 

分 类 号：S852.61[农业科学—基础兽医学]