纳米金标记核酸探针检测小反刍兽疫病毒核酸的研究  

Study on Peste Des Petits Ruminants Virus Nucleic Acid Detection Based on Gold-nanoparticle Conjuncted Probe

作　　者：陶春爱[1] 邱文英[1] 李刚[1] 田康乐[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所动物营养学国家重点实验室农业部兽用药物与兽医生物技术北京科学观测实验站,北京100193

出　　处：《中国生物工程杂志》2012年第7期89-94,共6页China Biotechnology

基　　金：国家自然科学基金(31172342);国家"863"计划(2008AA10Z411);国家公益性行业科研专项(200903037);"十一五"国家科技支撑计划(2006BAD6A13-4);FAO/IAEA项目(14133;515)资助项目

摘　　要：试验旨在探讨利用纳米金标记寡核苷酸探针快速检测小反刍兽疫病毒核酸的方法。针对小反刍兽疫病毒N基因的高度保守区设计两条特异寡核苷酸探针,一条探针5'端修饰生物素,另一条探针3'端修饰巯基。巯基化的探针通过Au-S键连接到纳米金颗粒上。靶核酸两端分别与两条探针结合,形成"生物素化探针-靶核酸-纳米金探针"复合物,该复合物通过生物素-亲和素系统,固定在固相载体上,最后银染放大信号。通过肉眼观察法、光镜观察法、分光光度法分析银染灰度,从而间接测定靶核酸的量。初步检测PPRV核酸最低浓度达10fmol/L,所需时间约为1.5h。该方法灵敏度高、操作简单,为临床检测小反刍兽疫病毒核酸提供实验数据和技术支持。In order to investigate a rapid method of detecting peste des petits ruminants virus(PPRV) with gold-nanoparticle conjuncted probe,two sets of specific oligonucleotide probes complementary to the ends of the highly conserved region of the PPRV nucleoprotein gene were designed.One was biotin-modified,the other was thioled and conjuncted to gold nanoparticles.During the course of hybridization,the two labeled probes binded to the target nucleic acid respectively,forming a 'sandwich'type complex,which was amplified by silver staining after incubated to streptavidin coated immuno-Microwells.Concentration of the target nucleic acid was determined by visual light microscope and spectrophotometry observation.The established method took around 1.5 h and the detection limit for nucleic acid was 10 fmol/L.It would provide theoretical and technical support for the clinical detection of PPRV since it is easy to operate and highly sensitive.

关 键 词：纳米金 PPRV 核酸探针 银染 

分 类 号：Q78[生物学—分子生物学]