摘要
目的建立布鲁氏菌的随机扩增DNA多态性的优化反应体系,用于布鲁氏菌分子流行病学研究。方法利用几种随机引物及其组合(P1、P2、P3、P4、P5、OPLO4、P3/5、P4/5)进行随机引物PCR的反应(AP-PCR,又称RAPD),及重复片断引物ERIC1R/ERIC2的随机扩增多态性;另外针对优选引物P5和OPLO4,分别对Mg2+浓度、dNTPs浓度、模板DNA浓度、引物的浓度及扩增过程中的退火温度等影响反应结果的因素,进行了一系列优化组合方案的试验及系统分析。结果引物P5、OPLO4和引物组合P4/5可以得到布鲁氏菌的高分辨率的分型带谱,并建立相应的优化反应体系。结论在严格的优化反应条件下,建立简单、快捷、灵敏度高的RAPD方法,用于布鲁氏菌的DNA多态性研究,为布鲁氏菌分子流行病学的深入研究提供资料。
To establish an optimal system of random amplified polymorphic DNA-PCR (AP-PCR, or RAPD) for the detection of Brucella, several combinations of the random primers (P_(1-2), P_(3-5), OPLP_4, P_(3/5), P_(4/5)) were to be used in the AP-PCR assay, and several factors that could effect the AP-PCR reaction, such as concentrations of Mg^(2+), dTNPs and template DNA ,concentration of primers as well as annealing temperature in the course of amplification, were investigated. A series of comparative experiments was carried to optimize the AP-PCR reaction, in which it was proved that the primers P_5, OPLO_4 and primer combination P_(4/5) were efficient to distinguish the types of Brucella species, thus establishing the optimized RAPD system. This optimized system is suitable for DNA polymorphism studies and important for the further studies in Brucella species.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2005年第6期482-485,共4页
Chinese Journal of Zoonoses
基金
国家973项目基金资助(NO.2002CB513201)
