摘要
针对猪流行性腹泻病毒(PEDV)的M基因设计LAMP引物,对反应体系的条件优化并进行特异性及敏感性等试验。结果显示:该方法在60℃下恒温扩增60min,使PEDV的M基因得到了高效率特异性扩增。本试验方法特异性好,与猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRSSV)、猪圆环病毒2型(PCV-2)、猪细小病毒(PPV)及猪伪狂犬病病毒(PRV)等无交叉反应,同时也具有较好的灵敏性,最低检测分子拷贝数为1.0×102 copies/μL。反应结束后加入SYBR GreenⅠ在紫外灯下观察颜色变化,结果显示阳性扩增产物呈现绿色荧光。结果表明:该LAMP检测方法特异性强,灵敏度高,操作便捷,适于临床检测PEDV。
The objective of the present study was to develop a loop-mediated isothermal amplifica- tion （LAMP） assay for detection of porcine epidemic diarrhea virus （PEDV）. I.AMP primers were designed based on the M gene of PEDV,optimizing the condition of reaction system and testing specificity and sensitivity. PEDV M gene could be specifically and efficiently amplified at 60℃ for 60 rain. The method was no cross reaction to swine fever virus （CSFV）,porcine reproductive and respiratory syndrome virus （PRSSV）, porcine circovirus type 2 （PCV-2）, porcine parvovirus （PPV） and pig pseudorabies virus （PRV）. Its minimum detection molecule copy number was 1.0×10^2 copies/μL. Reaction after joining SYBR Green I was observed color change under UV light,positive amplification product green showed fluorescence. The results showed that this LAMP assay was sensitive,specific,and easy to preform which could he applied to the rapid detec tion of PEDV infecton.
作者
王伟
张双翔
金志强
周碧君
文明
程振涛
冯旭芳
张海
唐宇
王开功
WANG Wei ZHANG Shuang-xiang JIN Zhi-qiang ZHOU Bi-jun WEN Ming CHENG Zhen-tao FENG Xu-fang ZHANG Hai TANG Yu WANG Kai gong(College of Animal Science ,Guizhou University, Guiyang 550025, China Guizhou Provincial Center lot Animal Disease Control and Prevention, Guiyang 550001, China Guiyang Agricultural Committee , Guiyang 550008, China The Pig Quality and Safety Pigs Engineering Technology Research Center, Guiyang 550025, China Guizhou Province Station Jot Livestocte Genetic Resources Man agem ent , Guiyang 550001, China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2017年第7期1220-1224,共5页
Chinese Journal of Veterinary Science
基金
贵州省科学技术厅农业攻关资助项目(黔科合NY[2015]3009-1号)
贵州省生猪质量安全工程技术研究中心建设资助项目(黔科合农C字[2011]4022号)
贵州省动物疫病防控与兽医公共卫生保障科技创新人才团队资助项目(黔科合人才团队[2015]4016号)
贵州省农业委员会科技计划资助项目(2015-01号)
