[1]郑新添,李晓华,胡晓丹,等.基于外膜蛋白Omp16的副猪嗜血杆菌抗体间接ELISA方法的建立[J].中国预防兽医学报,2018,(07):607-610.[doi:0.3969/j.issn.1008-0589.201710013]
 ZHENG Xin-tian,LI Xiao-hua,HU Xiao-dan,et al.Establishment of an indirect ELISA for detection of antibodies against the Omp16 of Haemophilus parasuis[J].Chinese journal of preventive veterinary medicine,2018,(07):607-610.[doi:0.3969/j.issn.1008-0589.201710013]
点击复制

基于外膜蛋白Omp16的副猪嗜血杆菌抗体间接ELISA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:
2018年07
页码:
607-610
栏目:
诊断和检测技术
出版日期:
2018-08-15

文章信息/Info

Title:
Establishment of an indirect ELISA for detection of antibodies against the Omp16 of Haemophilus parasuis
文章编号:
1008-0589(2018)7-0607-04
作者:
郑新添12李晓华12胡晓丹1许君茹1林炜明12杨小燕12*
(1. 龙岩学院 生命科学学院,福建 龙岩364012;2. 福建省生猪疫病防控工程技术研究中心,福建 龙岩364012)
Author(s):
ZHENG Xin-tian12 LI Xiao-hua12 HU Xiao-dan1 XU Jun-ru1 LIN Wei-ming12 YANG Xiao-yan12*
(1. College of Life Sciences, Longyan University, Longyan 364012, China;
2. Fujian Provincial Engineering & Technology Research Center for the Prevention and Control of Swine Diseases, Longyan 364012, China)
关键词:
副猪嗜血杆菌间接ELISAOmp16诊断
Keywords:
Haemophilus parasuis? indirect ELISA? Omp16? diagnosis
分类号:
S852.61
DOI:
0.3969/j.issn.1008-0589.201710013
文献标志码:
A
摘要:
为建立检测副猪嗜血杆菌(H.parasuis)抗体方法,本研究以H.parasuis外膜蛋白Omp16为包被抗原建立了检测H.parasuis的间接ELISA方法。反应条件经优化为:抗原包被浓度为1 μg/mL,血清稀释度为1:80;血清与抗原最佳作用时间为40 min,二抗稀释10 000倍,与一抗孵育30 min;TMB作用时间为30 min。利用该ELISA方法检测H.parasuis、胸膜肺炎放线杆菌、大肠杆菌和猪链球菌2型等血清,除H.parasuis为阳性外其余均为阴性,表明其特异性强。该ELISA方法可检测到最高稀释度为1∶512的阳性血清,敏感性高。重复性试验显示,其批内和批间的重复试验变异系数(CV)分别在3.62 %~8.97 %和4.3 %~9.50 %。该ELISA检测方法与进口ELISA试剂盒的阳性符合率、阴性符合率和总体符合率分别为87.8 %,90.7 %和88.6 %。本研究建立的间接ELISA方法,为H.parasuis血清流行病学调查及疫苗免疫评价等提供了重要手段。
Abstract:
An indirect ELISA for detection of antibody against Haemophilus parasuis was established using outer membrane protein Omp16 of H.parasuis as coating antigen. The reaction conditions of this assay were optimized, including: (a) coating antigen at 1μg/mL, (b) serum sample at 1:80 dilution, (c) serum incubated with antigen for 40min, (d) HRP-conjugated goat anti-pig IgG antibody at 1:10,000 dilution and incubated with serum for 30 min, (e) TMB incubated for 30 min. The ELISA was highly specific and showed no cross-reaction with the positive antiserum of other porcine disease. This ELISA was highly sensitive as it was able to detect at least H.parasuis antibody in 1:512 diluted serum. This assay was also repeatability with the inter samples coefficient of variation of raw optical density values for known positive samples in different runs was <10% (ranged from 3.62% to 8.97%), while intra sample coefficient of variation ranged from 4.3% to 9.50% between runs. The assay was validated by comparison with a commercial ELISA kit. The results showed that the positive coincident rate of this assay and commercial ELISA assay was 87.8%, and negative coincident rate was 90.7% and the total coincidence rate was 88.6%. The established ELISA kit could be a useful tool for detection of H.parasuis antibody, which can be used in serological epidemiology investigation.

参考文献/References:

[1] Oliveira S, Pijoan C. Haemophilus parasuis: new trends on diagnosis, epidemiology and control [J]. Vet Microbiol, 2004, 99: 1-12.
[2]王迪,朱瑞良,李兵,等. 2011年~2013年山东地区规模化养猪场猪体携带病原调查与分析[J]. 中国预防兽医学报,2015,37(07):557-560.
[3]Kavanova L, Prodelalova J, Nedbalcova K, et al. Immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in vitro [J]. Vet Microbiol, 2015, 180: 28-35.
[4]李仕新,黄彩梅,陈国开,等. 副猪嗜血杆菌检测技术的研究进展[J]. 中国动物检疫,2011,28(03):78-80.
[5]于新友,李天芝,苗立中,等. 副猪嗜血杆菌病诊断方法研究进展[J]. 广东畜牧兽医科技,2015,(01):8-11.
[6]Howell K J, Weinert L A, Chaudhuri R R, et al. The use of genome wide association methods to investigate pathogenicity, population structure and serovar in Haemophilus parasuis [J]. BMC Genomics, 2014, 15(01): 1179.
[7]Zheng Xin-tian, Yang Xiao-yan, Li Xiao-hua, et al. Omp16- based vaccine encapsulated by alginate-chitosan microspheres provides significant protection against Haemophilus parasuis in mice[J]. Vaccine, 2017, 35(10): 1417-1423.
[8]马福利,王丽荣,董永军,等. 副猪嗜血杆菌抗体间接ELISA检测方法的建立及应用[J]. 西北农业学报,2015,(05):29-33.
[9]刘双红,李大鹏,徐胜奎,等. 副猪嗜血杆菌间接ELISA检测方法的建立[J]. 中国预防兽医学报,2016,38(07):576-579.
[10]车勇良,陈如敬,江斌,等. 副猪嗜血杆菌oppA基因的克隆、表达及间接ELISA检测方法的建立[J]. 福建农林大学学报(自然科学版),2015,(03):282-288.
[11]张斌,汤承,岳华. 副猪嗜血杆菌主要外膜蛋白基因研究进展[J]. 动物医学进展,2013,(01):91-96.
[12]逄凤娇,俞正玉,何孔旺,等. 猪丁型冠状病毒重组N蛋白间接ELISA抗体检测方法的建立[J]. 中国预防兽医学报,2017,(06):461-465.
[13]王璐. 牛病毒性腹泻病毒E0基因的表达及间接ELISA检测方法的建立[D]. 乌鲁木齐:新疆农业大学,2012.
[14]孙东波,冯力,时洪艳,等. 猪传染性胃肠炎病毒重组N蛋白抗原间接ELISA抗体检测方法的建立[J]. 中国预防兽医学报,2006,28(05):572-576.
[15]Torremorell M,樊福好. 猪群监测:血清学的变异性、准确性和可靠性[J]. 动物科学与动物医学,2005,(02):20-21.
(本文编辑:李 爽)

相似文献/References:

[1]闫虹光,曲连东,刘家森,等.鸭病毒性肠炎间接ELISA诊断方法的建立及应用[J].中国预防兽医学报,2008,(09):721.
[2]曹竹婷,杨少华,杨宏军,等.检测牛轮状病毒抗体间接ELISA方法的建立[J].中国预防兽医学报,2009,(03):213.
 CAO Zhu-ting,YANG Shao-hua,YANG Hong-jun,et al.Establishment of indirect ELISA to detect bovine rotavirus antibody [J].Chinese journal of preventive veterinary medicine,2009,(07):213.
[3]宋 帅,林 彤,邵军军,等.O型口蹄疫病毒单克隆抗体的制备和鉴定[J].中国预防兽医学报,2009,(04):325.
 SONG Shuai,LIN Tong,SHAO Jun-jun,et al.Preparation and characterization of monoclonal antibody against Foot-and-mouth disease virus serotype O[J].Chinese journal of preventive veterinary medicine,2009,(07):325.
[4]姜 骞,李慕瑶,刘家森,等.犬细小病毒ELISA抗体检测试剂盒的研制及应用[J].中国预防兽医学报,2009,(02):141.
 JIANG Qian,LI Mu-yao,LIU Jia-sen,et al.Development and application of an indirect-ELISA kit for detection antibodies to canine parvovirus[J].Chinese journal of preventive veterinary medicine,2009,(07):141.
[5]王春仁,李志莲,翟延庆,等.奶牛新孢子虫重组蛋白NcSRS2t间接ELISA方法的建立及初步应用[J].中国预防兽医学报,2009,(02):145.
 WANG Chun-ren,LI Zhi-lian,ZHAI Yan-qing,et al.Development and preliminary application of indirect ELISA diagnostic method for Neospora caninum in dairy cattle[J].Chinese journal of preventive veterinary medicine,2009,(07):145.
[6]李军星,姜 平*,李玉峰,等.副猪嗜血杆菌热休克蛋白70基因的序列分析及抗原性鉴定 [J].中国预防兽医学报,2009,(07):519.
 LI Jun-xing,JIANG Ping*,LI Yu-feng,et al.Sequencing and expression of the HSP70 gene of Haemophilus parasuis and antigenicity of heat shock protein 70 [J].Chinese journal of preventive veterinary medicine,2009,(07):519.
[7]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,(07):618.
[8]徐 敏,王淑杰,蔡雪辉*,等.猪链球菌重组GDH蛋白间接ELISA检测方法的建立 [J].中国预防兽医学报,2009,(08):623.
 XU Min,WANG Shu-jie,CAI Xue-hui*,et al.Development of an indirect-ELISA for detection of antibodies against Streptococcus suis using recombinant GDH protein as antigen [J].Chinese journal of preventive veterinary medicine,2009,(07):623.
[9]陈 涛,赵建军,张海玲,等.水貂肠炎病毒VP2基因的原核表达及间接ELISA方法的建立 [J].中国预防兽医学报,2009,(09):712.
 CHEN Tao,ZHAO Jian-jun,ZHANG Hai-ling,et al.Prokaryotic expression of mink enteritis virus VP2gene and establishment of indirect ELISA[J].Chinese journal of preventive veterinary medicine,2009,(07):712.
[10]朱春红#,吴 娟#,张伟娟,等.肠炎沙门氏菌SEFA基因表达和间接ELISA检测方法的初步建立[J].中国预防兽医学报,2010,(01):44.
 ZHU Chun-hong#,WU Juan#,ZHANG Wei-juan,et al.Expression of subunit sefA of S. enteritidis SEF14 fimbriae and establishment of an indirect ELISA[J].Chinese journal of preventive veterinary medicine,2010,(07):44.

更新日期/Last Update: 2018-08-15