口蹄疫病毒核酸试纸条检测方法的建立

doi:10.16431/j.cnki.1671-7236.2015.09.011

《中国畜牧兽医》---唯一指定的官方网站 ›› 2015, Vol. 42 ›› Issue (9): 2292-2297.doi: 10.16431/j.cnki.1671-7236.2015.09.011

口蹄疫病毒核酸试纸条检测方法的建立

龚真莉¹, 蒋韬¹, 祁淑芸¹, 陈国栋², 刘艳红¹, 李勇¹

1. 中国农业科学院兰州兽医研究所, 家畜疫病病原生物学国家重点实验室, 农业部畜禽病毒学重点开放实验室, 兰州 730046;
2. 中农威特生物科技股份有限公司, 兰州 730046

收稿日期:2015-02-02 出版日期:2015-09-20 发布日期:2015-09-25
通讯作者: 蒋韬 E-mail:jiangtao@caas.cn
作者简介:龚真莉(1981-),女,甘肃兰州人,硕士,助理研究员,研究方向:病毒分子生物学,E-mail:gongzhenli@caas.cn
基金资助:
甘肃省科技重大专项计划项目(1002NKDA037);甘肃省农业科技成果转化资金计划(1305NCNA143)

Development of Nucleic Acid Lateral Flow Immunoassay Detection Method for Foot-and-mouth Disease Virus

GONG Zhen-li¹, JIANG Tao¹, QI Shu-yun¹, CHEN Guo-dong², LIU Yan-hong¹, LI Yong¹

1. Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. China Agricultural Vet Biological Science and Technlolgy Co., Ltd., Lanzhou 730046, China

Received:2015-02-02 Online:2015-09-20 Published:2015-09-25

摘要/Abstract

摘要： 本研究通过核酸探针与免疫层析相结合的方法,建立了一种简单、敏感、特异的检测口蹄疫病毒的方法。试验利用RT-PCR方法获得口蹄疫病毒3D核苷酸片段,在引物中设计了灵敏度高、特异性好的核酸探针——生物素和地高辛,使扩增产物结合探针。利用胶体金的放大原理将链霉亲和素与金颗粒形成胶体金混合物,从而与RT-PCR产物中的生物素探针结合。硝酸纤维素膜上端标记生物素化山羊抗兔IgG作为指控条带,下端标记抗地高辛抗体以捕获RT-PCR产物中的地高辛探针。组装金标试纸条,检测RT-PCR产物,结果表明该核酸试纸条可以检测到 0.3×10^-3~3×10^-3 μg/μL,敏感性高于琼脂糖凝胶电泳,两种方法的符合率高,核酸试纸条检测方法是一种敏感性高、成本低且费时短的新型检测方法。该方法为口蹄疫病毒检测提供了一种新的思路。

关键词: 口蹄疫病毒; 3D基因; 核酸试纸条; 生物素探针; 地高辛探针

Abstract: In this article, through the combination of nucleic acid probes and immune chromatography, a simple, sensitive and specific detection system——nucleic acid lateral flow immunoassay (NALFIA) for amplifing foot-and-mouth disease virus (FMDV) 3D RT-PCR products was established.An ultrasensitive nucleic acid biosensor (NAB) based on streptavidin-labeled gold nanoparticles dual labels and lateral flow strip biosensor (LFSB) were used in this system.The biotinylated goat anti-rabbit IgG was marked to the NC membrane as the alleged strip and the anti-digoxin antibody was labeled to the NC membrane to capture the digoxin probe.After assemblying gold-labeled strip and detecting RT-PCR products, the detection limit of NALFIA was 0.3×10^-3 to 3×10^-3μg/μL.The NALFIA was compared with agar gel electrophoresis analysis, the results showed that the sensitivity of NALFIA was higher than agar gel electrophoresis.There was an excellent agreement between the two methods.NALFIA was a method with high sensitive, low cost and short time.In conclusion, this method provided a good alternative to detect FMDV.

Key words: foot-and-mouth disease virus (FMDV); 3D gene; nucleic acid lateral flow immunoassay (NALFIA); biotin probe; digoxin probe

中图分类号:

龚真莉, 蒋韬, 祁淑芸, 陈国栋, 刘艳红, 李勇. 口蹄疫病毒核酸试纸条检测方法的建立[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2015, 42(9): 2292-2297.

GONG Zhen-li, JIANG Tao, QI Shu-yun, CHEN Guo-dong, LIU Yan-hong, LI Yong. Development of Nucleic Acid Lateral Flow Immunoassay Detection Method for Foot-and-mouth Disease Virus[J]. , 2015, 42(9): 2292-2297.

参考文献

[1] 朱彩珠,张强,卢永干.口蹄疫现状与未来[M].北京:中国农业科学技术出版社,2009.

[2] Tam S,Clavijo A,Engelhard E K,et al.Fluorescence-based multiplex Real-time RT-PCR arrays for the detection and serotype determination of foot-and-mouth disease virus[J].Journal of Virological Methods,2009,161(2):183-191.

[3] Yang S Z,Yang J F,Zhang G P,et al.Development of an immunochromatographic strip for the detection of antibodies against foot-and-mouth disease virus serotype O[J].Journal of Virological Methods,2010,165(2):139-144.

[4] Peng F H,Wang Z,Zhang S H,et al.Development of an immunochromatographic strip for rapid detection of H9 subtype avian influenza viruses[J].Clinical and Vaccine Immunology,2008,15(3):569-574.

[5] Frens G.Controlled nucleation for the regulation of particles size in monodisperse gold suspension[J].Nat Phys Sci,1973,241(105):20-21.

[6] Jiang T,Liang Z,Ren W W,et al.A simple and rapid colloidal gold-based immunochromatogarpic strip test for detection of FMDV serotype A[J].Virology Sinica,2011,26(1):30-39.

[7] 冯婷婷,刘一兵,侯惠仁,等.氟喹诺酮类药物胶体金试纸的研制[J].中国畜牧兽医,2012,39(9):54-58.

[8] Carter D J,Cary R B.Lateral flow microarrays:A novel platform for rapid nucleic acid detection based on miniaturized lateral flow chromatography[J].Nucleic Acids Research,2007,35(10):e74.

[9] Mens P F,de Bes H M,Sondo P,et al.Direct blood PCR in combination with nucleic acid lateral flow immunoassay for detection of Plasmodium species in settings where malaria is endemic[J].Journal of Clinical Microbiology,2012,50(11):3520-3525.

[10] de Koning M,Quint W,Struijk L,et al.Evaluation of a novel highly sensitive,broad-spectrum PCR-reverse hybridization assay for detection and identification of beta-papillomavirus DNA[J].Journal of Clinical Microbiology,2006,44(5):1792-1800.

[11] He Q,Zhang S Q,Zhang X B,et al.Ultrasensitive nucleic acid biosensor based on enzyme-gold nanoparticle dual label and lateral flow strip biosensor[J].Biosensors and Bioelectronics,2011,26(5):2018-2024.

[12] Mens P F,van Amerongen A,Sawa P,et al.Molecular diagnosis of malaria in the field:Development of a novel 1-step nucleic acid lateral flow immunoassay for the detection of all 4 human Plasmodium spp.and its evaluation in Mbita,Kenya[J].Diagnostic Microbiology and Infectious Disease,2008,61(4):421-427.

[13] 徐岱.PCR试纸条检测乙肝病毒DNA新方法研究[J].医学研究杂志,2006,35(8):102-103.

[14] 曹先维,张鹤龄.生物素标记核酸探针的分子杂交技术及其应用[J].病毒学,1991,6(1):1-14.

[15] 王义琴,孙勇如.生物素-亲合素系统及其应用[J].高技术通讯,2000,3:95-97.

[16] Dukes J P,King D P,Alexandersen S.Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus[J].Archives of Virology,2006,151(6):1093-1106.

口蹄疫病毒核酸试纸条检测方法的建立

Development of Nucleic Acid Lateral Flow Immunoassay Detection Method for Foot-and-mouth Disease Virus

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