牛流行热病毒可视化RT-LAMP检测技术的建立及应用
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广东省科技计划(2015B020203005,2015A020224039);广东省现代农业科技创新联盟建设项目(2016LM2150);广州市珠江科技新星专项(201610010073)


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

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  • 收稿日期:2016-06-27
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  • 在线发布日期: 2017-05-04