摘要
口蹄疫和水泡性口炎是牛常见高度急性病毒传染病,在全球范围内广泛存在。本研究旨在建立一种可同时鉴别口蹄疫病毒和水泡性口炎病毒的二重荧光RT-LAMP检测方法。根据口蹄疫病毒(FMDV)3D基因和水泡性口炎病毒(VSV)N基因的保守序列,设计了2套特异性引物,在每条内引物的5′端标记荧光基团,通过扩增产物颜色判断检测结果。优化反应条件,建立了可同时检测口蹄疫病毒和水泡性口炎病毒的二重荧光RT-LAMP方法。结果显示,该方法灵敏性高,每个反应最低能够检测到100个拷贝混合模板;特异性好,能在同一个反应管里检测到两种病毒,对其他牛病原体无扩增;干扰性小,扩增效率不受模板浓度影响。本研究建立的口蹄疫病毒和水泡性口炎病毒二重荧光RT-LAMP方法具有简便、快速、特异、敏感等优点,可用于FMDV和VSV的临床检测和流行病学调查。
Foot-and-mouth disease and vesicular stomatitis are serious cute disease common in cattle,and prevalence in large parts of the word.The aim of this study was to establish a duplex fluorescence-based reverse transcript loop-mediated isothermal amplification assay for detection of foot-and-mouth disease virus（FMDV）and vesicular stomatitis virus（VSV）.Two sets of specific primers were designed according to the conserved sequences of FMDV 3D gene and VSV Ngene,and the inner primers were synthesized with two different fluorophores at its 5′ends.A duplex fluorescence-based reverse transcript loop-mediated isothermal amplification assay was developed and used for detection of FMDV and VSV.The results showed that this assay could detect at least 100 copies of premixed plasmid containing the targets gene of FMDV and VSV per tube.The specificity of this assay was high,and be able to detect FMDV and VSV without any cross-reactions with other known non-targeted bovine pathogens.Different concentration template of FMDV and VSV could be identified when mixed together without any interference.These findings indicate that this duplex RT-LAMP assay is a simple,rapid,specific,and sensitive method for detection of FMDV and VSV,and can be applied in clinical detection and epidemiological investigation of FMDV and VSV.
作者
范晴
谢芝勋
谢志勤
谢丽基
黄娇玲
张艳芳
曾婷婷
王盛
罗思思
邓显文
刘加波
FAN Qing;XIE Zhi-xun;XIE Zhi-qin;XIE Li-ji;HUANG Jiao-ling;ZHANG Yan-fang;ZENG Ting-ting;WANG Sheng;LUO Si-si;DENG Xian-wen;LIU Jia-bo(Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2018年第5期996-1004,共9页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
广西科技重大专项(桂科AA17204057)
关键词
口蹄疫病毒
水泡性口炎病毒
二重荧光RT-LAMP
检测
foot-and-mouth disease virus
vesicular stomatitis virus
duplex fluorescence-based RT-LAMP
detection
