环介导恒温扩增技术快速检测对虾白斑综合征病毒方法的建立  

Establishment of Loop-mediated Isothermal Amplification for Detection of White spot syndrome virus

作　　者：李明云[1] 丁文超[1] 陈炯[1] 史雨红[1] 

机构地区：[1]宁波大学应用海洋生物技术教育部重点实验室,宁波315211

出　　处：《农业生物技术学报》2011年第1期186-190,共5页Journal of Agricultural Biotechnology

基　　金：长江学者和创新团队发展计划(No.IRT0734);国家科技支撑计划(No.2007BAD43B0);浙江省科技厅重点项目(No.2008C22049)资助

摘　　要：DNA环介导恒温扩增(loop-mediated isothermal amplification,LAMP)技术是一种特异、灵敏、快速的新型基因检测技术,其检测结果可以通过琼脂糖凝胶电泳观察,也可以通过加入核酸染料SYBR GreenⅠ进行肉眼观察。本研究根据对虾白斑综合征病毒(White spot syndrome virus,WSSV)的结构蛋白VP26基因序列,设计一套引物,成功建立了针对WSSV的LAMP检测方法。应用该方法,在65℃温育1h的条件下快速扩增病毒基因组DNA,琼脂糖凝胶电泳得到特异性梯度条带;在反应体系中添加SYBR GreenⅠ染料后,可通过肉眼观察有无荧光直接判定结果。该研究建立的LAMP法能特异性检出WSSV,其检测下限比PCR法低一个数量级,相当于0.563pg/μL的质粒(pMD19-T-VP26)浓度。表明所建立的WSSV LAMP检测法特异性好、灵敏度高、操作简单方便,有望作为WSSV快速诊断的常规方法。Loop-mediated isothermal amplification（LAMP） method is a novel gene detection technique with specificity,sensitive and rapidity,the products of which can be electrophoresed in 1.5% agarose gels,or visually inspected by adding nucleic acid dye（SYBR GreenⅠ）. In this paper,a one step loop-mediated isothermal amplification（LAMP） assay was developed for detection of White spot syndrome virus（WSSV）. A set of primers were designed from the VP26 gene sequence of WSSV. The assay was established to amplify WSSV genomic DNA by incubation at 65℃ for only 1 h,and required only a simple water bath or heating block to provide a constant temperature of 65℃. LAMP amplification products had a ladder-like appearance when electrophoresed on an agarose gel. With the addition of SYBR Green I,the presence of the target DNA could be detected by naked eyes. The detection limit of plasmid DNA （pMD19-T-VP26） by LAMP was 0.563 pg/μL which was found to be lower than that of the commonly used PCR method. The results indicate the suitability and simplicity of the test as a rapid,field diagnostic tool for WSSV.

关 键 词：对虾白斑综合征病毒 VP26基因 环介导恒温基因扩增检测技术 

分 类 号：S945.1[农业科学—水产养殖]