Study on rapid detection of double- stranded DNA viruses by the Triplex real- time PCR assay
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摘要: 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。
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关键词:
- 斑点叉尾鮰病毒 /
- 锦鲤疱疹病毒 /
- 流行性造血器官坏死病毒 /
- 三重荧光定量PCR
Abstract: 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 102copy·μL- 1for 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.-
Keywords:
- CCV /
- KHV /
- EHNV /
- Triplex real-time PCR
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