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

作　　者：贾坤[1] 韩太光 远立国[1] 孙凌霜[1] 宁章勇[1] 王衡[1] 李守军[1] 

机构地区：[1]华南农业大学兽医学院/广东省兽医临床重大疾病综合防控重点实验室/广东省宠物工程技术研究中心,广东广州510642

出　　处：《华南农业大学学报》2017年第3期15-20,共6页Journal of South China Agricultural University

基　　金：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)

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)、d NTPs mix浓度为1.2μmol·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。【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 Mg^2＋,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.

关 键 词：牛流行热 病毒检测 RT—LAMP PCR 灵敏性 特异性 

分 类 号：S854[农业科学—临床兽医学] S855[农业科学—兽医学]