华南农业大学学报2017，Vol.38Issue(3)：15-20,6.DOI:10.7671/j.issn.1001-411X.2017.03.003
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
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.关键词
牛流行热/病毒检测/RT-LAMP/PCR/灵敏性/特异性Key words
bovine ephemeral fever/virus detection/RT-LAMP/PCR/sensitivity/specificity分类
农业科技引用本文复制引用
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):15-20,6.基金项目
广东省科技计划(2015B020203005,2015A020224039) （2015B020203005,2015A020224039）
广东省现代农业科技创新联盟建设项目(2016LM2150) （2016LM2150）
广州市珠江科技新星专项(201610010073) （201610010073）
