适合早期诊断的非洲猪瘟sIgA抗体量子点免疫层析检测方法建立

doi:10.11843/j.issn.0366-6964.2023.10.027

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (10): 4311-4319.doi: 10.11843/j.issn.0366-6964.2023.10.027

适合早期诊断的非洲猪瘟sIgA抗体量子点免疫层析检测方法建立

谢青云^1,4, 易玮婕², 李嘉豪², 白昀^1,4, 谢星^1,4, 袁厅^1,4, 张越², 冯余凡², 赵东明³, 步志高³, 刘斐^2*, 冯志新^1,4*

1. 江苏省农业科学院兽医研究所, 农业农村部兽用生物制品工程重点实验室, 南京 210014;
2. 南京农业大学动物医学院单分子纳米生物学实验室, 南京 210095;
3. 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室, 哈尔滨 150069;
4. 兽用生物制品(泰州)国泰技术创新中心, 泰州 225300

收稿日期:2023-02-20 出版日期:2023-10-23 发布日期:2023-10-26
通讯作者: 冯志新,主要从事动物支原体病原学及猪呼吸道病的诊断与防控技术研究,E-mail:fzxjaas@163.com;刘斐,主要从事动物免疫及疫病防控技术研究,E-mail:feiliu24@njau.edu.cn
作者简介:谢青云(1990-),女,安徽宣城人,助理研究员,博士,主要从事抗体诊断试剂研究,E-mail:xqy816626@163.com;Tel:025-84390880;易玮婕(1997-),女,福建福州人,硕士生,主要从事动物疫病现场快速检测研究,E-mail:381779044@qq.com。
基金资助:
国家重点研发计划政府间国际科技创新合作重点专项（2019YFE0107300）；江苏省农业科技自主创新项目（CX（20）2035）

Development of Quantum Dot Microsphere-based Immunostrip for Early Detection of Specific sIgA Antibody to African Swine Fever Virus

XIE Qingyun^1,4, YI Weijie², LI Jiahao², BAI Yun^1,4, XIE Xing^1,4, YUAN Ting^1,4, ZHANG Yue², FENG Yufan², ZHAO Dongming³, BU Zhigao³, LIU Fei^2*, FENG Zhixin^1,4*

1. Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing 210014, China;
2. College of Veterinary Medicine, Nanjing Agricultural University, Single Molecule Nanometry Laboratory (Sinmolab), Nanjing 210095, China;
3. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China;
4. GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China

Received:2023-02-20 Online:2023-10-23 Published:2023-10-26

摘要/Abstract

摘要： 非洲猪瘟（African swine fever，ASF）是由非洲猪瘟病毒感染引起的一种烈性猪传染病，其极高的传染性和发病率给养猪产业造成了巨大的经济损失。目前尚无安全有效的ASF疫苗，因此早期监测是防控ASF最重要的解决方案之一。建立敏感、快速的ASF现场即时检验方法，对维护生猪产业繁荣具有重要意义。首先基于氨基和羧基的偶联反应制备量子点微球（quantum dot microsphere，QDM）偶联ASFV重组抗原蛋白p30的检测探针和QDM-兔抗鸡IgY质控探针；接着在检测线和质控线分别固定鼠抗猪IgA-Sc片段抗体和鸡IgY蛋白制备侧向流免疫试纸条；待检动物口腔液中的ASF sIgA抗体经QDM-p30捕获可被固定至T线。将ASFV阳性口腔液以2倍梯度稀释，检测该方法的灵敏度。通过检测其他几种常见猪病病毒验证该方法的特异性。通过检测ASFV阴性和阳性动物的口腔液临床样本评价该方法的临床诊断效能。该方法特异性好，与PRV、CSFV、PRRV不存在交叉反应；灵敏度高，最低检出稀释度可达1：64；耗时短，可在20 min内完成检测；操作便捷，无需侵入式采样；检出时间早，人工感染最早可于感染后第6天检出。本研究开发了一种口腔液中ASF sIgA抗体的现场即时检验（point-of-care testing，POCT）量子点免疫试纸条，为ASFV的监测和防控提供了新的技术支持和补充。

关键词: 非洲猪瘟, sIgA抗体, 量子点免疫试纸条, 现场即时检验

Abstract: African swine fever (ASF), caused by the African swine fever virus (ASFV), is a devastating infectious disease in wild boars and domestic pigs. The extremely high infectivity and morbidity of ASFV have caused enormous socioeconomic losses. Given that there is no effective vaccine available for ASFV infection, early diagnosis is crucial for the prevention and control of ASF. Therefore, it is of great significance to develop a sensitive and rapid method for the on-site detection of ASFV. Firstly, the detection probe of quantum dot microspheres (QDM) coupled ASFV recombinant antigen p30 and the quality control probe of QDM coupled anti-chicken IgY were prepared based on the condensation reaction between amino and carboxyl groups. Then, the immunostrip was prepared by immobilized anti-porcine IgA-Sc antibody and chicken IgY protein on the detection line and control line, respectively. The ASFV-specific sIgA antibody in the oral fluid can be captured by the QDM-p30 fixed on the T-line. Finally, the detection sensitivity was tested by detecting the ASFV-positive oral fluid with double gradient dilution; the specificity was verified by detecting several other common swine disease viruses; and the clinical diagnostic efficacy was evaluated by testing clinical oral fluid samples from ASFV-negative and ASFV-positive animals. The proposed method shows good specificity and no cross-reaction with PRV, CSFV and PRRV; shows high sensitivity with the lowest detected dilution of 1:64 for ASFV-positive oral fluid; shows short time consuming within 20 minutes; and is easy to operate without intrusive sampling. In addition, sIgA-positive conversion could be detected as early as the 6th day after ASFV infection, suggesting the potential for early diagnosis. Here, we developed a QDM-based immunostrip for on-site early detection of ASFV sIgA antibody in oral fluid, which provides new technical support and supplement for ASFV monitoring, prevention and control.

Key words: African swine fever, sIgA antibody, QDMs-based immunostrip, point-of care testing

中图分类号:

S858.285.3

谢青云, 易玮婕, 李嘉豪, 白昀, 谢星, 袁厅, 张越, 冯余凡, 赵东明, 步志高, 刘斐, 冯志新. 适合早期诊断的非洲猪瘟sIgA抗体量子点免疫层析检测方法建立[J]. 畜牧兽医学报, 2023, 54(10): 4311-4319.

XIE Qingyun, YI Weijie, LI Jiahao, BAI Yun, XIE Xing, YUAN Ting, ZHANG Yue, FENG Yufan, ZHAO Dongming, BU Zhigao, LIU Fei, FENG Zhixin. Development of Quantum Dot Microsphere-based Immunostrip for Early Detection of Specific sIgA Antibody to African Swine Fever Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4311-4319.

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适合早期诊断的非洲猪瘟sIgA抗体量子点免疫层析检测方法建立

Development of Quantum Dot Microsphere-based Immunostrip for Early Detection of Specific sIgA Antibody to African Swine Fever Virus

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