建立环介导等温扩增可视化检测方法用于检测中国针对经典猪瘟的 HCLV 疫苗。
Development of a loop-mediated isothermal amplification for visual detection of the HCLV vaccine against classical swine fever in China.
机构信息
Division of Swine Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
出版信息
J Virol Methods. 2011 Jan;171(1):200-5. doi: 10.1016/j.jviromet.2010.10.025. Epub 2010 Nov 3.
A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the rapid and specific detection of HCLV vaccine strain against classical swine fever. Four primers were designed for amplification of NS5B gene region with Bst DNA polymerase at a constant temperature of 65°C. The products showed ladder-like pattern on 2% agarose gel, and can be visualised after addition of SYBR Green I dye. The detection limit of the assay was 5 copies of the HCLV genome per reaction. No cross-reaction with other porcine viruses including different wild-type CSFV strains and the bovine viral diarrhoea virus was observed. The agreement between the LAMP and TaqMan real-time RT-PCR assays was 94.4% for the detection of 72 batches of HCLV vaccine. The assay provides a rapid tool for the control of vaccine quality and can be an accompanying assay of the LAMP for wild-type CSFV described previously for differential diagnosis.
建立并评价了一种环介导等温扩增（LAMP）检测方法，用于快速、特异性检测猪瘟兔化弱毒疫苗株。该方法针对 NS5B 基因区域设计了 4 条引物，使用 Bst DNA 聚合酶在 65°C 恒温条件下进行扩增。产物在 2%琼脂糖凝胶上呈现梯状条带，添加 SYBR Green I 染料后可进行可视化观察。该检测方法的检测限为每个反应 5 个 HCLV 基因组拷贝。与其他猪病毒如不同野生型猪瘟病毒株和牛病毒性腹泻病毒无交叉反应。LAMP 与 TaqMan 实时 RT-PCR 检测方法对 72 批 HCLV 疫苗检测的符合率为 94.4%。该检测方法为疫苗质量控制提供了一种快速工具，并且可以与先前描述的用于鉴别诊断的野生型猪瘟 LAMP 检测方法配套使用。
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