牛传染性胸膜肺炎抗体间接ELISA检测方法的建立

doi:10.11843/j.issn.0366-6964.2020.02.014

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (2): 329-336.doi: 10.11843/j.issn.0366-6964.2020.02.014

牛传染性胸膜肺炎抗体间接ELISA检测方法的建立

郝文君¹, 李媛¹, 周效乾², 王秀梅^1*, 辛九庆^1*

1. 中国农业科学院哈尔滨兽医研究所动物支原体创新团队, 哈尔滨 150069;
2. 东北农业大学动物医学学院, 哈尔滨 150030

收稿日期:2019-08-15 出版日期:2020-02-23 发布日期:2020-02-22
通讯作者: 辛九庆,主要从事动物传染病及其病原分子流行病学研究,E-mail:xinjiuqing2001@126.com;王秀梅,主要从事动物传染病及其病原分子流行病学研究,E-mail:xmwang99@163.com
作者简介:郝文君(1995-),女,山东淄博人,硕士生,主要从事动物支原体致病机理和诊断方法的研究
基金资助:
"十三五"国家重点研发计划（2016YFD0500900）

Establishment of Indirect ELISA for Detecting Antibody to Contagious Bovine Pleuropneumonia

HAO Wenjun¹, LI Yuan¹, ZHOU Xiaoqian², WANG Xiumei^1*, XIN Jiuqing^1*

1. Animal Mycoplasma Innovation Team, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China;
2. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China

Received:2019-08-15 Online:2020-02-23 Published:2020-02-22

摘要/Abstract

摘要： 旨在建立一种检测牛传染性胸膜肺炎（CBPP）血清抗体的间接ELISA方法，用于该病的监测。利用生物信息学软件对CBPP的病原丝状支原体丝状亚种国内分离株Ben-1株的全基因组进行分析，选取脂蛋白rP0308作为包被抗原，通过一系列反应条件的优化，建立了基于rP0308蛋白的间接ELISA抗体检测方法，并对其性能进行评价。结果显示该方法的敏感性为92%，特异性为96%，与牛支原体、牛鼻支原体、无乳支原体、口蹄疫、牛病毒性腹泻、牛传染性鼻气管炎和牛结核分枝杆菌等阳性血清均不发生交叉反应，批内变异系数在2.41%~6.03%，批间变异系数在2.94%~6.59%，重复性良好。利用本方法和商品化的cELISA试剂盒分别对实验室保存的1 648份临床样品进行检测，该方法与商品化试剂盒的阴性符合率为89.1%，阳性符合率为79.2%，总符合率为88.7%。以上研究结果表明，本研究建立的间接ELISA方法具有良好的敏感性、特异性和重复性，具有良好的临床应用前景。

关键词: 牛传染性胸膜肺炎, rP0308, 间接ELISA

Abstract: An indirect ELISA was developed for detecting antibodies to Mycoplasma mycoides subsp. mycoides SC (MmmSC), the agent of contagious bovine pleuropneumonia (CBPP), and used for CBPP surveillance. Based on bioinformatics assay of the whole genome sequence of MmmSC isolates Ben-1, lipoprotein rP0308 was selected as coating antigen. An indirect ELISA was established after optimization of a series of conditions, and its performance was evaluated. As a result, the diagnostic sensitivity and specificity of the indirect ELISA were 92% and 96%, respectively. The ELISA did not react with other antibodies against Mycoplasma bovis, Mycoplasma bovirhinis, Mycoplasma agalactiae, foot and mouth disease, bovine infectious rhinotracheitis, Mycobacterium tuberculosis. Reproducibility analysis revealed that the coefficients of variation of samples within and between runs were 2.41%-6.03%, and 2.94%-6.59%, respectively. One thousand six hundred and forty-eight serum samples from our lab were tested using this ELISA system and a commercial kit, a total of 1 405/1 576 (89.1%) of the sera found positive in the commercial kit were also positive in the ELISA, and 57/72 (79.2%) of the sera found negative in the both system. The overall coincidence was 88.7%. The above results suggested that the established ELISA demonstrated high specificity, sensitivity and reproducibility, and could be a promising tool applied in the field.

Key words: contagious bovine pleuropneumonia, rP0308, indirect ELISA

中图分类号:

S852.62

郝文君, 李媛, 周效乾, 王秀梅, 辛九庆. 牛传染性胸膜肺炎抗体间接ELISA检测方法的建立[J]. 畜牧兽医学报, 2020, 51(2): 329-336.

HAO Wenjun, LI Yuan, ZHOU Xiaoqian, WANG Xiumei, XIN Jiuqing. Establishment of Indirect ELISA for Detecting Antibody to Contagious Bovine Pleuropneumonia[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(2): 329-336.

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牛传染性胸膜肺炎抗体间接ELISA检测方法的建立

Establishment of Indirect ELISA for Detecting Antibody to Contagious Bovine Pleuropneumonia

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