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2025年8月20日 星期三
  2018, Vol. 26 Issue (2): 330-338    
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
口蹄疫病毒O、A和AsiaⅠ型分型基因芯片的构建与评价
刘志鹏1,欧阳达1,黄小波2,2,滑翔3,赵玉佳3,曹三杰4,文翼平3,伍锐3,赵勤1,文心田4
1. 四川农业大学
2. 四川农业大学动物医学院
3. 四川农业大学 动物医学院/动物传染病与基因芯片实验室
4. 四川农业大学动物医学院动物传染病与基因芯片实验室
Construction of a Gene Chip for Differentiating Serotypes O, A and AsiaⅠ of Foot-and-mouth disease virus
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摘要 口蹄疫(Foot-and-mouth disease, FMD)是由口蹄疫病毒(Foot-and-mouth disease virus, FMDV)引起的一种高度接触传染性动物疫病。本研究拟建立一种能区分FMDV O/A/AsiaⅠ型的基因芯片。根据FMDV O/A/AsiaⅠ型的VP1基因序列设计3对特异性引物,用RT-PCR扩增获得248、206和239 bp的三个靶基因片段,并针对这三个片段分别设计了3种探针,以引物荧光标记法标记靶基因,建立了一种鉴别FMDV O/A/AsiaⅠ型的分型基因芯片并进行了敏感性、特异性、重复性和保存期评价。结果表明最佳反应条件为:水化温度37 ℃,封闭液为0.25% BH4Na/25% 乙醇,杂交温度为42 ℃。灵敏性实验表明,FMDV-O 检测限为118 pg/mL,FMDV-A为18.4 pg/mL,FMDV-AsiaⅠ为129 pg/mL,芯片方法灵敏度比常规RT-PCR高10~100倍;特异性实验表明,O、A和AsiaⅠ三型之间无交叉杂交;该芯片可至少保存3个月仍能重复使用。本研究建立的FMDV分型基因芯片为O、A和AsiaⅠ型鉴别提供了新方法。
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刘志鹏
欧阳达
黄小波
滑翔
赵玉佳
曹三杰
文翼平
伍锐
赵勤
文心田
关键词 FMD病毒基因芯片分型鉴别构建    
Abstract:Foot-and-mouth disease (FMD) is an acute, hot, and highly contagious animal disease caused by Foot-and-mouth disease virus (FMDV). This study is to establish a gene chip that distinguishes FMDV serotypes O, A and AsiaⅠ. Three specific primers were designed according to the VP1 gene sequences of FMDV, and three target fragments of 248, 206 and 239 bp were obtained by RT-PCR. Three kinds of probes were designed and labeled with primer markers. A genotyping microarray encoding O, A and AsiaⅠ was established and its sensitivity, specificity, reproducibility and shelf life were evaluated. The optimum reaction conditions were as follows: hydration temperature was 37 ℃, confluent concentration was 0.25% BH4Na, 25% ethanol, and hybridization temperature was 42 ℃. Sensitivity test showed that FMDV-O, FMDV-A and FMDV-AsiaⅠ were 10-5, 10-7, 10-5 after the band is not visible. The PCR detection limits were: FMDV-O 118 pg/mL, 4.3×106 copies/μL; FMDV-A 18.4 pg/mL, 8.1×104 copies/μL; FMDV-AsiaⅠ 129 pg/mL, 4.9×105 copies/μL. The FMDV-O dilution reaches 10-6; the FMDV-A dilution reaches 10-8; the FMDV-AsiaⅠ dilution reaches 10-6, the sensitivity of the method was 10~100 times higher than that of a conventional PCR. There was no cross-hybrid among the detecting surfaces O, A and A1; the chip was reproducible and the prepared chip could be stored for at least three months. In this study, the experimental conditions were optimized, and a fast and simple FMDV classification method was established to provide a new technique for the classification of FMDV.
Key wordsFoot-and-mouth disease virus    Microarray    Typing    Screening    Construction
收稿日期: 2017-07-17      出版日期: 2018-02-04
ZTFLH:  S852.659  
基金资助:国家重点研发计划(2016YFD0500700)-猪重要疫病的诊断与检测新技术研究
通讯作者: 文心田     E-mail: xintian3211@126.com
引用本文:   
刘志鹏 欧阳达 黄小波 滑翔 赵玉佳 曹三杰 文翼平 伍锐 赵勤 文心田. 口蹄疫病毒O、A和AsiaⅠ型分型基因芯片的构建与评价[J]. , 2018, 26(2): 330-338.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2018/V26/I2/330
 
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