检测鸡毒支原体抗体的间接ELISA方法和HI试验方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2023.05.027

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (5): 2062-2072.doi: 10.11843/j.issn.0366-6964.2023.05.027

检测鸡毒支原体抗体的间接ELISA方法和HI试验方法的建立及初步应用

陈杨¹, 孟林春¹, 郭梦娇¹, 张成成¹, 薄宗义², 楚电峰³, 曹永忠², 吴艳涛^1,2, 张小荣^1*

1. 扬州大学兽医学院江苏省动物重要疫病预防控制协同创新中心, 扬州 225009;
2. 扬州大学农业科技发展研究院教育部农业与农产品安全国际合作联合实验室, 扬州 225009;
3. 青岛易邦生物工程有限公司动物基因工程疫苗国家重点实验室, 青岛 266114

收稿日期:2022-09-13 出版日期:2023-05-23 发布日期:2023-05-20
通讯作者: 张小荣,主要从事动物传染病学研究,E-mail:zxr@yzu.edu.cn
作者简介:陈杨(1997-),男,江苏南通人,博士生,主要从事动物传染病学研究,E-mail:953679181@qq.com;孟林春(1995-),女,山西大同人,硕士生,主要从事动物传染病学研究,E-mail:2366714405@qq.com
基金资助:
财政部和农业农村部:国家现代农业产业技术体系资助项目(CARS-40-K16);国家重点研发计划"科技助力经济2020"重点专项(SQ2020YFF0426460);动物基因工程疫苗国家重点实验室开放课题(AGVSKL-ZD-202003);江苏高校优势学科建设工程资助项目(2018年)

Establishment and Preliminary Application of Indirect ELISA Method and HI Test for Detection of Mycoplasma Gallisepticum Antibody

CHEN Yang¹, MENG Linchun¹, GUO Mengjiao¹, ZHANG Chengcheng¹, BO Zongyi², CHU Dianfeng³, CAO Yongzhong², WU Yantao^1,2, ZHANG Xiaorong^1*

1. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
2. International Research Laboratory of Agriculture and Agri-Product Safety, Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou 225009, China;
3. State Key Laboratory of Genetically Engineered Veterinary Vaccines, Qingdao Yebio Biological Engineering Co., Ltd, Qingdao 266114, China

Received:2022-09-13 Online:2023-05-23 Published:2023-05-20

摘要/Abstract

摘要： 本研究旨在建立一种检测鸡毒支原体(Mycoplasma gallisepticum,MG)抗体的间接ELISA方法,结合HI试验,为MG感染的监测和净化提供一套有效的组合技术方案。以原核表达的VlhA 3.03重组蛋白(rVlhA)作为包被抗原,使用单一稀释法构建了关于血清抗体滴度与1∶500血清稀释度处S/P值的回归方程lg(抗体滴度)=1.257×lg(1∶500处S/P值)＋3.709,R²=0.917 6,S/P临界值为0.32,临界滴度是1 200。经验证,rVlhA-ELISA方法具有良好的特异性、重复性,最低能检出 1∶2 000 稀释的阳性血清。选择国内MG分离株SH/2020-1作为血凝抑制抗原建立HI试验方法。使用rVlhA-ELISA方法联合HI试验进行血清样品检测,IDEXX-MG方法作为对照,借助RT-qPCR方法监测MG感染情况。结果表明rVlhA-ELISA方法和IDEXX-MG监测的血清抗体趋势与HI复核结果均一致;rVlhA-ELISA与HI联合判定结果与IDEXX-MG符合率可达91.53%。上述结果显示,本研究建立的rVlhA-ELISA方法和HI试验具有较好的临床应用价值,可以初步应用于MG感染的临床大规模、快速筛查并对结果进行复核。

关键词: 鸡毒支原体, VlhA蛋白, 抗体检测, 间接ELISA

Abstract: This study aimed at establishing an indirect-ELISA method for the titer detection of Mycoplasma gallisepticum (MG) antibodies in chicken, which could provide a set of effective technology for large-scale clinical monitoring of MG infection combined with HI test established. Prokaryotic-expressed protein VlhA3.03 (rVlhA) was obtained as envelope antigen. The linear equation lg (antibody titer)=1.257×lg (S/P value at 1:500) +3.709 of serum antibody titer and S/P value at 1:500 was established by a single serum dilution test and the correlation coefficient (R²) was 0.917 6. The cutoff value of negative and positive S/P was determined to be 0.32, and the corresponding titer is 1 200. The rVlhA-ELISA showed good specificity and repeatability. The detection limit of MG positive sera is 1:2 000. An HI assay was also established using domestic MG isolate SH/2020-1 as hemagglutination inhibition test antigen. The combined rVlhA-ELISA method and HI test, and IDEXX-MG were compared in serum sample detection, with RT-qPCR method to monitor MG infection. The results showed that the trend of serum antibody monitored by rVlhA-ELISA and IDEXX-MG was consistent with the result of HI assay. The coincidence rate between combined rVlhA-ELISA and HI, and IDEXX-MG was 91.53%. These results indicated that the rVlhA-ELISA and HI test established in this study were beneficial to clinical application, which can be applied to large-scale and rapid clinical screening of MG infection and recheck the results.

Key words: Mycoplasma gallisepticum, variable lipoprotein haemagglutinin, antibody detection, indirect enzyme-linked immunosorbent assay

中图分类号:

S852.62

陈杨, 孟林春, 郭梦娇, 张成成, 薄宗义, 楚电峰, 曹永忠, 吴艳涛, 张小荣. 检测鸡毒支原体抗体的间接ELISA方法和HI试验方法的建立及初步应用[J]. 畜牧兽医学报, 2023, 54(5): 2062-2072.

CHEN Yang, MENG Linchun, GUO Mengjiao, ZHANG Chengcheng, BO Zongyi, CHU Dianfeng, CAO Yongzhong, WU Yantao, ZHANG Xiaorong. Establishment and Preliminary Application of Indirect ELISA Method and HI Test for Detection of Mycoplasma Gallisepticum Antibody[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2062-2072.

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检测鸡毒支原体抗体的间接ELISA方法和HI试验方法的建立及初步应用

Establishment and Preliminary Application of Indirect ELISA Method and HI Test for Detection of Mycoplasma Gallisepticum Antibody

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