纳米金标记核酸探针检测小反刍兽疫 病毒核酸的研究

中国生物工程杂志

2012, Vol. 32

Issue (07): 89-94

技术与方法

纳米金标记核酸探针检测小反刍兽疫病毒核酸的研究

陶春爱, 邱文英, 李刚, 田康乐

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

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

TAO Chun-ai, QIU Wen-ying, LI Gang, TIAN Kang-le

State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences/ Beijing Scientific Observation and Experiment Station for Veterinary Drug and Veterinary Biotechnology, Ministry of Agriculture, Beijing 100193, China

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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。

关键词： 纳米金; PPRV; 核酸探针; 银染

Abstract: 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.

Key words: Gold nanoparticle PPRV Oligonucleotide probes Silver staining

收稿日期: 2011-12-21 出版日期: 2012-07-25

ZTFLH:

Q78

基金资助: 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

通讯作者: 李刚 E-mail: gli358@gmail.com

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引用本文:

陶春爱, 邱文英, 李刚, 田康乐. 纳米金标记核酸探针检测小反刍兽疫病毒核酸的研究[J]. 中国生物工程杂志, 2012, 32(07): 89-94.

TAO Chun-ai, QIU Wen-ying, LI Gang, TIAN Kang-le. Study on Peste Des Petits Ruminants Virus Nucleic Acid Detection Based on Gold-nanoparticle Conjuncted Probe. China Biotechnology, 2012, 32(07): 89-94.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I07/89

[1] Libeau G, Prehaud C, Lancelot R, et al. Development of a competitive ELISA for detecting antibodies to the peste des petits ruminants virus using a recombinant nucleoprotein. Res Vet Sci, 1995,58 (1): 50-55.
[2] Li L, Bao J, Wu X, et al. Rapid detection of peste des petits ruminants virus by a reverse transcription loop-mediated isothermal amplification assay. J Virol Methods, 2010,170 (1-2): 37-41.
[3] Powell A C, Paciotti G F, Libutti S K. Colloidal gold: a novel nanoparticle for targeted cancer therapeutics. Methods Mol Biol, 2010,624: 375-384.
[4] Mirkin C A, Letsinger R L, Mucic R C, et al. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature, 1996,382 (6592): 607-609.
[5] Nam J M, Thaxton C S, Mirkin C A. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science, 2003,301 (5641): 1884-1886.
[6] Georganopoulou D G, Chang L, Nam J M, et al. Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer’s disease. Proc Natl Acad Sci U S A. 2005,102 (7): 2273-2276.
[7] 赵立凡, 李柏生, 黑笑涵, 等.银染增强的纳米金探针技术检测微量核酸. 中国生物化学与分子生物学报. 2006 (11): 919-923. Zhao L F,Li B S, Hei X H, et al. A colorimetric polynucleotides detection method based on nanoparticle probes with silver staining enhancement.Chinese Journal of Biochemistry and Molecular Biology,2006, (11): 919-923.
[8] Yang M, Wang G. Label-free immunosensor based on gold nanoparticle silver enhancement. Anal Biochem, 2009,385 (1): 128-131.
[9] Wang Y F, Pang D W, Zhang Z L, et al. Visual gene diagnosis of HBV and HCV based on nanoparticle probe amplification and silver staining enhancement. J Med Virol, 2003,70 (2): 205-211.
[10] Kangatharalingam N. Essenberg, M. Modified pyrogallol-initiated immunogold-silver enhancement technique applicable to prokaryotes. J Microbiol Methods, 2000,41 (3): 211-217.
[11] Hacker G W, Graf A H, Thurner J. Application of silver acetate autometallography in histopathology: a new detection method for use in immunogold silver staining, lectin histochemistry and in situ hybridization. Verh Dtsch Ges Pathol, 1990,74: 368-372.
[12] 张鑫宇, 孙怀昌, 刘文俊, 等. 非洲猪瘟病毒p72 基因的纳米金探针研究. 扬州大学学报(农业与生命科学版), 2011,32 (1): 55-58. Zhang X Y,Sun H C,Liu W J,et al.Study on the nanogold probe to p72 gene of African swine fever virus.Journal of Yangzhou University (Agricultural and Life Science Edition),2011,32 (1): 55-58.
[13] Jadzinsky P D, Calero G, Ackerson C J, et al. Structure of a thiol monolayer-protected gold nanoparticle at 1.1 A resolution. Science, 2007,318 (5849): 430-433.

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