大口黑鲈虹彩病毒重组酶聚合酶扩增结合侧流层析试纸条(RPA-LFD)快速诊断方法的建立与应用

Development of a recombinase polymerase amplification combined with lateral flow dipstick assay for visual detection of largemouth bass virus

  • 摘要:
    目的 建立一种快速灵敏、可视化的适用于临床样品检测大口黑鲈虹彩病毒(largemouth bass virus,LMBV)的方法。
    方法 本研究基于LMBV主要衣壳蛋白(MCP)基因序列设计特异性引物及探针,通过优化反应温度、反应时间等反应条件,建立结合重组酶聚合酶扩增技术(RPA)和侧流层析试纸条(LFD) (RPA-LFD)的快速可视化检测方法,并从敏感性、特异性及临床适用性等方面评价该方法的检测性能。
    结果 该方法在38 ℃恒温扩增10 min后即可通过侧流层析试纸条实现可视化判读,且与大口黑鲈其他主要病原无交叉反应。RPA-LFD反应最低检测限为10个拷贝LMBV/μL,比常规PCR检测的灵敏度高约10倍。此外,RPA-LFD方法和常规PCR方法在临床样本检测上高度一致(符合率97.2%)。
    结论 本研究建立的RPA-LFD方法为LMBV的现场诊断提供了一种快速、特异、灵敏及可视化的方法。

     

    Abstract: Largemouth bass (Micropterus salmoides) has become one of the important economical freshwater fish species in China in recent years. However, the frequent outbreak of viral diseases induced by largemouth bass virus (LMBV) always caused high mortality and led to heavy economic losses in the largemouth bass aquaculture industry. Notably, LMBV was also found to infect other species of Percidae, such as smallmouth bass (Micropterus dolomieu) and bluegill (Lepomis macrochirus). However, rapid, specific, and visual methods for LMBV detection were limited up to now. In this study, a rapid and convenient technique combining recombinase polymerase amplification (RPA) with lateral flow dipsticks (LFD) was established to detect LMBV. The specific, sensitive primers and probes targeting major capsid protein (MCP) of LMBV were screened. The assay of RPA-LFD took approximately 10 min at 38℃ using a water bath, and exhibited no cross-reactivity with several other aquatic pathogens. The detection limit of RPA-LFD was 10 copies/μL, which was 10 times more sensitive than that from routine PCR. Moreover, 36 tissue samples from largemouth bass were detected to validate the application of RPA-LFD. The coincidence rate of the positive test samples was 97.2% between RPA-LFD and PCR. Taken together, the established RPA-LFD assay here provided a rapid, convenient, specific and sensitive method for field diagnosis of LMBV without special equipment.

     

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