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    贾坤, 韩太光, 远立国, 孙凌霜, 宁章勇, 王衡, 李守军. 牛流行热病毒可视化RT-LAMP检测技术的建立及应用[J]. 华南农业大学学报, 2017, 38(3): 15-20. DOI: 10.7671/j.issn.1001-411X.2017.03.003
    引用本文: 贾坤, 韩太光, 远立国, 孙凌霜, 宁章勇, 王衡, 李守军. 牛流行热病毒可视化RT-LAMP检测技术的建立及应用[J]. 华南农业大学学报, 2017, 38(3): 15-20. DOI: 10.7671/j.issn.1001-411X.2017.03.003
    shu
    JIA Kun, HAN Taiguang, YUAN Liguo, SUN Lingshuang, NING Zhangyong, WANG Heng, LI Shoujun. Establishment and application of visual RT-LAMP technology for detecting bovine ephemeral fever virus[J]. Journal of South China Agricultural University, 2017, 38(3): 15-20. DOI: 10.7671/j.issn.1001-411X.2017.03.003
    Citation: JIA Kun, HAN Taiguang, YUAN Liguo, SUN Lingshuang, NING Zhangyong, WANG Heng, LI Shoujun. Establishment and application of visual RT-LAMP technology for detecting bovine ephemeral fever virus[J]. Journal of South China Agricultural University, 2017, 38(3): 15-20. DOI: 10.7671/j.issn.1001-411X.2017.03.003
    shu

    牛流行热病毒可视化RT-LAMP检测技术的建立及应用

    Establishment and application of visual RT-LAMP technology for detecting bovine ephemeral fever virus

      摘要
    • 摘要:
      目的  建立一种能够快速、简便、可视化地检测牛流行热病毒的分子生物学方法。
      方法  根据牛流行热病毒G蛋白基因的6个保守区域设计2对引物，建立牛流行热病毒可视化逆转录环介导等温扩增(Reverse transcription loop-mediated isothermal amplification，RT-LAMP)检测技术，优化RT-LAMP的反应条件，将其与PCR方法进行比较。
      结果  当Mg2+浓度为3 mmol·L-1、甜菜碱浓度为0.4 mol·L-1、dNTPs mix浓度为1.2 μmol·L-1、内外引物浓度比例为8:1、反应温度为63 ℃时, 反应梯形条带最明显，在反应40 min后可以观察到明显的梯形条带。建立的RT-LAMP检测方法特异性好，只对牛流行热病毒进行扩增；灵敏度比普通PCR高10倍。
      结论  该方法操作简便，特异性强，结果判读方便，可用于牛流行热的快速检测。

       

      Abstract:
      Objective  To develop a rapid, convenient and visual molecular assay for detecting bovine ephemeral fever virus (BEFV).
      Method  Two pairs of primers based on six conserved regions in G protein gene of BEFV were designed, and the visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting BEFV was developed. The reaction conditions of RT-LAMP assay were optimized, and the assay was compared with PCR.
      Result  The reaction ladder bands from the RT-LAMP assay were most obvious when the RT-LAMP reaction system contained 3 mmol·L-1 Mg2+, 0.4 mol·L-1 betaine and 1.2 μmol·L-1 dNTPs mix, the ratio of inner to outer primers was 8:1, and the reaction temperature was 63 ℃. Clear reaction ladder bands were observed after 40 min of reaction. The established RT-LAMP assay had excellent specificity with only BEFV being amplified. The sensitivity was 10 times higher than that of ordinary PCR.
      Conclusion  The visual RT-LAMP assay is easy to operate and highly specific, and the results can be conveniently determined. This method can be used for the rapid detection of bovine epidemic fever.

       

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