嗜水气单胞菌的随机扩增多态DNA分型  

Genetic Diversity of the Pathogen Aeromonas hydrophila by Random Amplified Polymorphic DNA

作　　者：卢强[1] 任瑞文[1] 胡岩 王文东[1] 郑伟[1] 

机构地区：[1]解放军军需大学动物科技系 [2]解放军77156部队医院防疫所

出　　处：《中国兽医学报》2002年第1期34-36,共3页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目 (3 0 0 70 5 87);军队医药卫生青年基金资助项目 (98Q0 78)

摘　　要：用 4 5条随机引物对不同来源的 9株嗜水气单胞菌进行了 DNA指纹分析 ,其中 10条引物呈现良好的多态性和稳定性。建立的随机扩增多态 DNA(RAPD)反应的最佳条件 :模板 DNA 2 0 ng,随机引物 0 .8μm ol/ L ,d NTP 15 0μmol/ L,Mg2 + 3.5 mm ol/ L,Taq酶 2 .0 U;95℃预变性 5 min,94℃变性 6 0 s,36℃退火 70 s,72℃延伸 12 0 s,4 0个循环。初步建立了嗜水气单胞菌的 RAPD指纹图谱 ,分析了 9株嗜水气单胞菌之间的遗传距离。据此将 9株菌归为 3个组 ,为该菌的分型提供了新的方法。Nine Aeromonas hydrophila recovered from aquatic animal were characterized by random amplified polymorphic DNA(RAPD),45 random primers were used,and 10 of them gained steady DNA fingerprints.The result suggested the optimization of the RAPD was performed by using 25 μL mixture containing 10 pg of template DNA,2 5 μL 10×buffer,3 5 μL of 25 μmol/L MgCl 2,1 5 μL of 20 μmol/L primer,1 5 μL of 2 5 μmol/L dNTP.PCR amplification was performed with a PTC 51B PCR system by using the following temperature program:1 cycle of denaturation for 5 min at 95℃;melting at 94℃ for 60 sec;annealing at 36℃ for 70 sec;and elongation at 72℃ for 120 sec;40 cycles;and a final extension round at 72℃ for 10 min.On the basis of RAPD,the DNA fingerprints of A.hydrophila were constructed.The results also showed that species A.hydrophila constituted a genetically heterogeneous group of strains,encompassing within an homogeneous or clonal lineage comprised solely of typical strains.The RAPD is proved to be a sensitive and reproducible tool for detecting and typing Aeromonas hydrophila.

关 键 词：PAPD 嗜水气单胞菌 细菌分型 

分 类 号：S852.61[农业科学—基础兽医学]