采用通用引物PCR配合SSCP和RFLP技术检测鱼病病原菌

Universal primer PCR with SSCP and RFLP for identification of fish disease pathogens

采用通用引物PCR(UPPCR)、PCR RFLP、PCR SSCP技术 ,研究快速鉴别鱼病病原菌的分子生物学诊断技术。结果发现 ,采用细菌 16SrRNA基因保守区特异性引物 ,以嗜水气单胞菌、鲁克氏耶尔森菌、鳗弧菌、柱状曲挠杆菌、乙型链球菌、荧光假单胞菌等部分常见鱼病病原菌为对象 ,可以建立一种UPPCR技术。该技术能在保证实验条件不变的基础上 ,检出上述所有细菌 ,并还可检出大肠杆菌和双歧杆菌等非鱼病病原菌。并且认为 ,该法与SSCP配合即采用UPPCR SSCP技术能较好地鉴别被检菌而用于鱼病病原菌的快速诊断。

A universal primer PCR(UPPCR), PCR RFLP and PCR SSCP had been screened for a molecular biological method that permitted the rapid identification of fish pathogens among them. The results showed that a universal primer PCR technology was available with specific primers from conserved regions of bacterial 16S ribosomal RNA genes. The bacteria tested included some common causative agents of fish diseases such as Aeromonas hydrophila, Yersinia ruckeri, Vibrio anguillarum, Flexibacter columnaris, beta Streptococcus, Pseudomonas fluorescens. The approach allowed the bacteria above and Escherichia coli and Bifidobacterium catenulatum detectable without any alterations of the experiment conditions. However, this study also found that the identification of species of bacteria tested depended on the combination of UPPCR and SSCP(PPCR SSCP), which was better than that of UPPCR and RFLP(UPPCR RFLP) and made rapid diagnosis of fish disease pathogens possible.