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根据哈维氏弧菌(Vibro harveyi)溶血素基因vhhP2的保守序列设计特异性引物,建立了SYBR Green I实时定量PCR检测哈维氏弧菌的方法。构建含vhhP2基因的重组质粒作为标准品,进行SYBR Green I实时定量PCR,在Tm为60℃时,扩增产物的熔解曲线仅有一个特异峰,扩增所得标准曲线为y=–3.331x+37.48,相关系数为0.998,扩增效率为1,最低可检测到7个拷贝。实验结果表明该检测技术具有较高的特异性、敏感性和重复性,对哈维氏弧菌病的快速诊断和流行病学调查有重要意义。
According to the conserved sequence of vhhP2 gene of Vibro harveyi hemolysin gene, specific primers were designed to detect Vibrio harveyi by SYBR Green I real-time PCR. The recombinant plasmids containing vhhP2 gene were constructed as standard and SYBR Green I real-time PCR was performed. When the Tm was 60 ℃, there was only one specific peak of the melting curve of the amplified product. The standard curve obtained was y = -3.331x + 37.48, the correlation coefficient was 0.998, the amplification efficiency was 1, the lowest detectable 7 copies. The experimental results show that the detection technique has high specificity, sensitivity and repeatability, and is of great significance for rapid diagnosis and epidemiological investigation of Vibrio harveyi.