鲑鱼甲病毒逆转录环介导等温扩增(RT-LAMP)检测方法研究
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S941.41+9

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国家质量监督检验检疫总局科技计划项目(2016IK013)


Development of a RT-LAMP Detection Method for Salmonid alphavirus
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    摘要:

    鲑鱼甲病毒(Salmonid alphavirus,SAV)是鲑鱼胰脏病(Salmon pancreas disease,PD)病原,主要感染鲑科鱼类,目前在我国尚未分离到。为提高出入境鱼类中的SAV检测效率,根据SAV nsP1基因片段,设计3对特异性引物,建立了检测SAV的逆转录环介导等温扩增(RT-LAMP)检测方法。该方法使用25 μL反应体系,经优化后的反应温度为65 ℃,反应时间为1 h,检测限可达10个拷贝数的病毒核酸,且不与传染性鲑鱼贫血病毒、病毒性出血性败血症病毒、传染性造血器官坏死病毒、病毒性神经坏死病病毒、草鱼呼肠孤病毒和鲤春病毒血症病毒的RNA产生交叉反应。在反应体系中加入染料后,反应结果肉眼直接可见。本研究建立的RT-LAMP方法是一种特异性强、方便快捷的SAV检测方法,适合SAV的现场初筛和核酸检测。

    Abstract:

    Salmonid alphavirus(SAV)is the pathogen of salmon pancreas disease(PD),mainly infects fishes in salmonidae family. At present,SAV hasn't been isolated in China yet. In order to improve the detection efficiency of SAV,3 pairs of specific primers were designed according to the nsP1 gene segment,and a reverse transcription mediated isothermal amplification(RT-LAMP)method for detecting SAV was established. The reaction system was 25 μL,the optimized reaction temperature was 65 ℃,and the reaction time was 1 h. The minimum detection limit was 10 copies of viral nucleic acids. Specificity tests showed that,the developed method didn't have any cross-reactions with other RNA viruses,including infectious salmon anaemia virus,viral haemorrhagic septicaemia virus,infectious haematopoietic necrosis virus,viral nervous necrosis,grass carp reovirus,and spring viraemia of carp virus. After the addition of dyes,the reaction results was directly visible to the naked eyes. In summary,a specific,convenient detection method was developed,and it was suitable for SAV primary screening and nucleic acid detection.

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刘艳华,任彤,谈艳苗,张利峰.鲑鱼甲病毒逆转录环介导等温扩增(RT-LAMP)检测方法研究[J].《中国动物检疫》编辑部,2018,35(10):99-103.

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  • 在线发布日期: 2018-10-11
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