[1]苗海生,李 乐*,廖德芳,等.蓝舌病病毒抗原捕获ELISA检测方法的建立[J].中国预防兽医学报,2015,(03):216-219.[doi:10.3969/j.issn.1008-0589.2015.03.13]
 MIAO Hai-sheng,LI Le*,LIAO De-fang,et al.Establishment of AC-ELISA for detection of bluetongue virus[J].Chinese journal of preventive veterinary medicine,2015,(03):216-219.[doi:10.3969/j.issn.1008-0589.2015.03.13]
点击复制

蓝舌病病毒抗原捕获ELISA检测方法的建立
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2015年03
页码:
216-219
栏目:
诊断和检测技术
出版日期:
2015-03-20

文章信息/Info

Title:
Establishment of AC-ELISA for detection of bluetongue virus
文章编号:
1008-0589(2015)03-0216-04
作者:
 苗海生李 乐*廖德芳朱建波寇美龄杨永钦孙 强李华春*
(云南省畜牧兽医科学院,云南 昆明650224)
Author(s):
 MIAO Hai-sheng LI Le* LIAO De-fang ZHU Jian-bo KOU Mei-ling YANG Yong-qinSUN Qiang LI Hua-chun*
(Yunnan Animal Science and Veterinary Institute, Kunming 650224, China)
关键词:
 蓝舌病病毒AC-ELISA抗原检测
Keywords:
 bluetongue virus AC-ELISA antigen detection
分类号:
S852.65
DOI:
10.3969/j.issn.1008-0589.2015.03.13
文献标志码:
A
摘要:
 为建立蓝舌病病毒(BTV)病原学检测方法,本研究以纯化的BTV-1免疫绵羊和兔子,制备高免血清。以绵羊抗BTV血清为捕捉抗体、以兔抗BTV血清和羊抗兔IgG-HRP为检测抗体建立了检测BTV的抗原捕获ELISA(AC-ELISA)方法。该方法能够特异性检测BTV,对羊痘病毒、赤羽病病毒无交叉反应,与鹿流行性出血热鸡胚样品有微量交叉反应,但样品OD450nm/阴性对照OD450nm低于阴阳性临界值,不影响结果判定;敏感性试验检测结果表明该方法可以检出102.79 TCID50的病毒;批内和批间重复性试验变异系数分别为1.44 %~8.46 %和2.26 %~12.44 %;采用该方法与RT-PCR方法对60份鸡胚样品进行检测,阳性符合率为90 %。本研究建立的AC-ELISA方法为BTV抗原检测提供了一种快速、实用的技术手段。
Abstract:
 To establish an assay for bluetongue virus (BTV) detection, the sheep and rabbit anti-serum against BTV were prepared with the purified BTV-1 replicated in BHK-21 cell to immune the sheep and rabbit, respectively, and the antigen capture ELISA (AC-ELISA) was then developed with the sheep anti-serum as coating antibody for antigen capture and with the rabbit antiserum for sandwich binding antigen following by IgG-HRP detection. Under the optimized reaction conditions, the AC-ELISA was high specificity and sensitivity for BTV detection with a limit detection of 102.79 TCID50, and no cross-reaction was found in goatpox virus, epizootic hemorrhagic disease virus and Akabane virus. In addition, the coefficient of variation for intra-assay and inter-assay were 1.44% to 8.46% and 2.26% to 12.44%. And the coincidence rate with RT-PCR was 90%. These results demonstrated the AC-ELISA was rapid and practical detection technology for BTV.

参考文献/References:

[1] Peter M, Matthew B, Philip A. Bluetongue [M]. London: Academic Press, 2008.
[2]Bhanuprakash V, Indrani B K, Hosamani M, et al. Bluetongue vaccines: the past, present and future [J]. Expert Rev Vaccines, 2009, 8(2): 191-204.
[3]Davies F, Mungai J, Pini A, et al. A new bluetongue virus serotype isolated in Kenya [J]. Vet Microbiol, 1992, 31(1): 25-32.
[4]Makoschey B, Beer M, Zientara S, et al. Bluetongue control-a new challenge for Europe [J]. Berl Munch Tierarztl Wochenschr, 2008, 121(7-8): 306-313.
[5]朱建波,杨仕标,李华春,等. 谨防以羊、牛为侵害对象的“杀手”-蓝舌病流行病学及防控技术研究进展[J]. 中国动物卫生保健,2008,107(1):105-108.
[6]Sotelo E, Llorente F, Rebollo B, et al. Development and evaluation of a new epitope-blocking ELISA for universal detection of antibodies to West Nile virus [J]. J Virol Methods, 2011, 174(1-2): 35-41.
[7]Franco O L, Ogas M L, Combessies G, et al. Single dilution Avidity-blocking ELISA as an alternative to the bovine viral diarrhea virus neutralization test [J]. J Virol Methods, 2011, 175(2): 228-235.
[8]Al M Z, Bruyas J F, Pellerin J L, et al. Evaluation of bluetongue virus (BTV) decontamination techniques for caprine embryos produced in vivo [J]. Theriogenology, 2012, 78(6): 1286- 1293.

相似文献/References:

[1]梁化春,齐景伟,刘淑英*,等.绵羊肺腺瘤病的病理学及RT-PCR诊断 [J].中国预防兽医学报,2009,(06):443.
 LIANG Hua-chun,QI Jing-wei,LIU Shu-ying*,et al.The pathological and RT-PCR diagnosis ofsheep pulmonary adenomatosis [J].Chinese journal of preventive veterinary medicine,2009,(03):443.
[2]刘合义,孙留霞,王进轶,等.牛冠状病毒重组N蛋白间接ELISA检测方法的建立 [J].中国预防兽医学报,2009,(08):618.
 LIU He-yi,SUN Liu-xia,WANG Jin-yi,et al.Development of an indirect ELISA for the detectionof Bovine coronavirus using recombinant N protein [J].Chinese journal of preventive veterinary medicine,2009,(03):618.
[3]宋红梅,杨 涛,马健男,等.蓝舌病病毒VP7基因的原核表达[J].中国预防兽医学报,2009,(12):925.
 SONG Hong-mei,YANG Tao,MA Jian-nan,et al.Prokaryotic expression of Bluetongue virus VP7 gene [J].Chinese journal of preventive veterinary medicine,2009,(03):925.
[4]秦永丽,孙恩成,耿宏伟,等.抗蓝舌病病毒VP7和NS2蛋白单克隆抗体的制备及鉴定[J].中国预防兽医学报,2011,(10):816.[doi:10.3969/j.issn.1008-0589.2011.10.15]
 QIN Yong-li,SUN En-cheng,GENG Hong-wei,et al.Production and identification of monoclonal antibodies against VP7 and NS2 of bluetongue virus[J].Chinese journal of preventive veterinary medicine,2011,(03):816.[doi:10.3969/j.issn.1008-0589.2011.10.15]
[5]耿宏伟,秦永丽,李俊平,等.蓝舌病病毒重组VP7蛋白单克隆抗体制备及竞争ELISA检测方法的建立[J].中国预防兽医学报,2012,(05):388.[doi:10.3969/j.issn.1008-0589.2012.05.12]
 GENG Hong-wei,QIN Yong-li,LI Jun-ping,et al.Development of monoclone antibody competitive ELISA for detection of Bluetongue virus antibodies[J].Chinese journal of preventive veterinary medicine,2012,(03):388.[doi:10.3969/j.issn.1008-0589.2012.05.12]
[6]魏 鹏,孙恩成,刘霓红,等.抗蓝舌病病毒VP6和VP7蛋白单克隆抗体的制备及鉴定[J].中国预防兽医学报,2012,(05):412.[doi:10.3969/j.issn.1008-0589.2012.05.18]
 WEI Peng,SUN En-cheng,LIU Ni-hong,et al.Preparation and identification of monoclonal antibodies against VP6 and VP7 of bluetongue virus[J].Chinese journal of preventive veterinary medicine,2012,(03):412.[doi:10.3969/j.issn.1008-0589.2012.05.18]
[7]魏 天,徐青元,耿宏伟,等.蓝舌病病毒VP7蛋白竞争抑制群特异性构象抗原表位的初步鉴定[J].中国预防兽医学报,2014,(01):63.[doi:10.3969/j.issn.1008-0589.2014.01.16]
 WEI Tian,XU Qing-yuan,GENG Hong-wei,et al.Identification of the group-specific conformational epitopein VP7 of bluetongue virus[J].Chinese journal of preventive veterinary medicine,2014,(03):63.[doi:10.3969/j.issn.1008-0589.2014.01.16]
[8]杨 涛,徐青元,孙恩成,等.蓝舌病病毒反向遗传操作系统的建立[J].中国预防兽医学报,2014,(02):85.[doi:10.3969/j.issn.1008-0589.2014.02.01]
 YANG Tao,XU Qing-yuan,SUN En-cheng,et al.Establishment of a reverse genetics system for bluetongue virus[J].Chinese journal of preventive veterinary medicine,2014,(03):85.[doi:10.3969/j.issn.1008-0589.2014.02.01]
[9]秦玉寅,王雪峰,韦华冕,等.马传染性贫血病毒弱毒疫苗gp90多样性及其对病毒体外增殖的影响[J].中国预防兽医学报,2014,(03):182.[doi:10.3969/j.issn.1008-0589.2014.03.04]
 QIN Yu-yin,WANG Xue-feng,WEI Hua-mian,et al.gp90 genetic diversity of EIAV attenuated vaccine and its impact on EIAV’s proliferation in vitro[J].Chinese journal of preventive veterinary medicine,2014,(03):182.[doi:10.3969/j.issn.1008-0589.2014.03.04]
[10]刘 畅,李 磊,于立新,等.稳定表达绵羊肺腺瘤病毒受体Hyal-2蛋白细胞系的建立[J].中国预防兽医学报,2014,(03):187.[doi:10.3969/j.issn.1008-0589.2014.03.05]
 LIU Chang,LI Lei,YU Li-xin,et al. Development of a cell line stably expressing Jaagsiekte retrovirus receptor Hyal-2 protein[J].Chinese journal of preventive veterinary medicine,2014,(03):187.[doi:10.3969/j.issn.1008-0589.2014.03.05]
[11]冯瑜菲,赵国辉,徐青元,等. 蓝舌病病毒一步RT-PCR检测方法的建立[J].中国预防兽医学报,2014,(09):712.[doi:10.3969/j.issn.1008-0589.2014.09.12]
 FENG Yu-fei,ZHAO Guo-hui,XU Qing-yuan,et al.Development of one-step RT-PCR method for the detection of bluetongue virus[J].Chinese journal of preventive veterinary medicine,2014,(03):712.[doi:10.3969/j.issn.1008-0589.2014.09.12]
[12]罗小暖,王 芳,崔玉东*,等.蓝舌病病毒VP2蛋白展示口蹄疫病毒中和表位的研究[J].中国预防兽医学报,2014,(09):731.[doi:10.3969/j.issn.1008-0589.2014.09.17]
 LUO Xiao-nuan,WANG Fang,CUI Yu-dong*,et al.Display expression of food-and-mouth disease virus neutralizing epitope on bluetongue virus VP2 protein[J].Chinese journal of preventive veterinary medicine,2014,(03):731.[doi:10.3969/j.issn.1008-0589.2014.09.17]

备注/Memo

备注/Memo:
收稿日期:2014-04-22
基金项目:国家农业公益性行业科研专项(201303035);公益性行业(农业)科研专项(201103008);云南省应用基础研究面上项目(2013FZ166)
作者简介:苗海生(1980-),男,山东金乡人,助理研究员,主要从事动物病毒学研究.
*通信作者:E-mail:lilesn@sina.comli_huachun@hotmail.com
更新日期/Last Update: 2015-04-14