猪瘟病毒RT-LAMP-LFD检测方法的建立与应用
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国家自然科学基金(U1405216, 31172321和31472200);广东省高等教育科技创新特殊项目(2012CXZD0013)


Rapid detection of classical swine fever virus by loop-mediatedisothermal amplification combined with lateral flow dipstick method
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    摘要:

    【目的】建立一种新型的、能在基层场地进行实时便捷诊断的猪瘟病毒检测方法。【方法】环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)-横向流动试纸条技术(Lateral flow dipstick, LFD)相结合,针对猪瘟病毒(Classical swine fever virus, CSFV) NS5B基因设计一套特异性引物及生物素标记探针,结合异硫氰酸荧光素标记的扩增产物,建立RT-LAMP-LFD检测方法。【结果】所建立的方法能够特异地检测出CSFV的存在,检测PRRSV、JEV、PCV-2等其他病毒均为阴性;所建立的CSFV-RT-LAMP 方法对RNA的最低检测限为50 pg;反应快速,整个扩增反应可在1 h 内完成;操作简单, 不需要PCR 仪等复杂的仪器,结果可经肉眼观察。【结论】应用RT-LAMP-LFD 检测CSFV特异性强、灵敏度高,并且操作安全、简便、快捷,可以满足基层检疫的需要。

    Abstract:

    【Objective】 The experiment was conducted to establish a rapid real time detection method of classical swine fever virus at local fields. 【Method】 A rapid detection method(RT-LAMP-LFD) of classical swine fever virus(CSFV) was developed by loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) method. The LAMP method for amplification of the CSFV NS5B gene using fluorescein isothiocyanate-labeled primer was combined with a chromatographic LFD for rapid and simple visual detection of CSFV. 【Result】 This method could specifically detect CSFV, but not other virus such as PRRSV, JEV and PCV-2. The minimum detection limit of the CSFV-RT-LAMP-LFD method was 50 pg. The detection could be finished within 1 h without using complex instruments such as PCR machine and the results were visible by eye. 【Conclusion】 This RT-LAMP-LFD method is a highly specific and sensitive, safe, rapid and simple way for field detection of CSFV infection.

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朱俊灵,叶佐东,邓洁汝,勾红潮,陈金顶.猪瘟病毒RT-LAMP-LFD检测方法的建立与应用[J].华南农业大学学报,2016,37(1):1-7

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  • 收稿日期:2015-04-13
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  • 在线发布日期: 2016-01-08