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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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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.
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Received: 21 December 2011
Published: 25 July 2012
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[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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