鱼源嗜水气单胞菌和维氏气单胞菌PCR-RFLP鉴别方法的建立

Establishment of PCR-RFLP Method for Discrimination of Aeromonas hydrophila and Aeromonas veronii Isolated from Diseased Fish

嗜水气单胞菌和维氏气单胞菌是鱼类常见条件致病菌,采用传统的形态分类和生理生化反应进行鉴别费时费力,有时还会错误判断,为了提高鉴别效率和准确率,有必要建立一种快速鉴别两种细菌的分子生物学方法。选取12株鱼源致病气单胞菌使用细菌16SrDNA通用引物扩增细菌目的片段,将所测得序列校正后构建系统发育树以明确菌种,并依据种间序列差异筛选合适的限制性核酸内切酶,对嗜水气单胞菌和维氏气单胞菌进行PCR-RFLP酶切图谱的鉴别分析。筛选出嗜水气单胞菌16SrDNA的特异性限制性核酸内切酶SnaBⅠ和可以鉴别维氏气单胞菌的限制性核酸内切酶BseNⅠ,建立了一种快速鉴别嗜水气单胞菌和维氏气单胞菌的PCR-RFLP方法。研究结果对于淡水养殖鱼类嗜水气单胞菌和维氏气单胞菌细菌性病害的病原快速鉴别具有参考和应用价值。

Aeromonas hydrophila and Aeromonas veronii were familiar opportunistic pathogens for fish.The discrimination of them using traditionary method such as morphological classification and physiological-biochemical identification was time-consuming and needed great energy,and they might result in erroneous identification sometimes.In order to improve efficiency and accuracy,it was necessary to establish a molecular biology method for rapid discrimination of them.Universal primer was used to amplify 16 S rDNA gene of 12 aeromonads isolated from diseased fish,and sequences acquired were applied to build phylogenetic tree to validate species of aeromonads.Suitable restriction endonucleases were chosen based on the different sequences between species,and PCR-RFLP analysis was utilized to discriminate the above two Aeromonas spp.Through experiments,Sna BⅠwas chosen to digest 16 S rDNA of A.hydrophila specifically and Bse NⅠwas chosen to discriminate A.veronii from A.hydrophila quickly.A PCR-RFLP method was established to rapid discrimination of A.hydrophila and A.veronii and it was of a certain reference significance and application value for the rapid discrimination of A.hydrophila and A.veronii in the bacterial diseases of freshwater fish.