三重荧光定量PCR快速检测水产品中的双链DNA病毒的研究

Study on rapid detection of double- stranded DNA viruses by the Triplex real- time PCR assay

目的:建立对水产品中三种双链DNA病毒(斑点叉尾鮰病毒、锦鲤疱疹病毒、流行性造血器官坏死病毒)快速、敏感、特异的检测方法。方法:针对三种病毒的DNA聚合酶基因的保守序列设计三对特异性引物和三条Taqman探针,优化体系,建立三重荧光定量PCR体系,并对该方法的灵敏性和特异性评估。结果:建立的三重荧光定量PCR体系特异性强,引物之间及引物和探针之间无相互干扰。对三种病毒的检测限均能达到102拷贝/μL。结论:该方法体系稳定,重复性好,可操作性强,对水产品中的双链DNA病毒的快速检测具有重要的应用价值。

Object： Establish rapid, sensitive and specific methods of detecting three double-strand DNA viruses, Channel catfish virus （ CCV）, Koi herpesvirus （ KHV）, Epizootic hematopoietic necrosis virus （ EHNV）, from fish samples.Method：Three pairs of specific primers and three Taqman probes were designed targeting for conserved sequence of DNA polymerase gene of three kinds of virus.The Triplex real-time PCR assay was established.The sensitivity and specificity of the method were evaluated.Results=The sensitivity of this method was 102 copy·μL-1 for detecting DNA of three viruses.The assay was specified for three kinds of virus CCV, KHV and EHNV. In the experiment, non-interference between primers and probes was found.Conclusion=The Triplex real-time PCR assay was a stable and reliable method for rapid detection of three double-strand DNA viruses in aquatic animal products.