施马伦贝格病毒RT-LAMP检测方法的建立  

Development of RT-LAMP for detecting Schmallenberg virus

作　　者：陈轩[1] 杨素[1] 黄海超[1] 王岚[1] 沙才华[1] 廖秀云[1] 徐海聂[1] 罗宝正[1] 薄清如[1] 

机构地区：[1]珠海出入境检验检疫局,广东珠海519015

出　　处：《中国兽医科学》2015年第8期826-831,共6页Chinese Veterinary Science

基　　金：国家质量监督检验检疫总局资助项目(2013IK050)

摘　　要：根据GenBank中施马伦贝格病毒S基因的保守区序列,设计1组对应目的片段6个区域的4条特异性引物(包括1对内引物和1对外引物)进行核酸扩增,建立RT-LAMP反应体系并进行反应条件优化,确定最佳内外引物浓度分别为1.2、0.15μmol/L,最佳反应条件为61℃保温60min。特异性和敏感性试验结果表明,本研究建立的施马伦贝格病毒RT-LAMP能检测到133 copies/μL的施马伦贝格病毒克隆质粒。结果表明,该RT-LAMP具有特异性强、灵敏度高和快速简便等优点,是在进出境口岸、养殖场或基层实验室等场所开展快速检测的良好方法。Applying reverse transcription loop-mediated isothermal amplification（RT-LAMP）,a set of four specific primers including one pair of inner primers and one pair of outer primers were successfully designed to recognize six distinct conserved regions of S segment gene of Sehmallenberg virus（SBV）. By optimizing reaction system and conditions, RT-LAMP method for detecting SBV was developed. The results indicated that the optimum concentration of inner primers and outer primers was 1.2 μmol/L and 0.15 μmol/L respectively;the optimum reaction temperature was verified to be 61 ℃ and the reaction time was 60 rain. The specificity and sensitivity of this method were examined and the detectable, minimum plasmid copy number was 133 copies/μL. The results indicated that RT-LAMP method is a simple,rapid,sensitive and specific method for detecting SBV, and it is a good alternative method for on-farm disease diagnosis or entry-exit quarantine of Sehmallenberg disease.

关 键 词：施马伦贝格病毒 RT-LAMP 检测 

分 类 号：S852.659.5[农业科学—基础兽医学]