猪流行性腹泻病毒荧光微球免疫层析抗原检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.09.035

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (9): 4572-4580.doi: 10.11843/j.issn.0366-6964.2025.09.035

猪流行性腹泻病毒荧光微球免疫层析抗原检测方法的建立

李慧敏^1,2, 雷铭楷^1,2, 阮胜男^1,2, 李盼盼¹, 李文涛^1,2,3*, 何启盖^1,2*

1. 华中农业大学动物医学院, 武汉 430070;
2. 农业微生物资源发掘与利用全国重点实验室, 武汉 430070;
3. 湖北洪山实验室, 武汉 430070

收稿日期:2024-11-27 发布日期:2025-09-30
通讯作者: 何启盖，主要从事猪传染病防控与净化研究，E-mail:he628@mail.hzau.edu.cn，李文涛，主要从事重大疫病的诊断技术、致病机制和新型疫苗的研究，E-mail:wentao@mail.hzau.edu.cn
作者简介:李慧敏(1997-)，女，吉林图们人，博士生，主要从事体外诊断试剂开发研究，E-mail：leehyemin@webmail.hzau.edu.cn；雷铭楷(1997-)，男，四川资阳人，博士生，主要从事猪病毒性腹泻综合防控技术研究，E-mail：mklei@webmail.hzau.edu.cn；
基金资助:
国家自然科学基金面上项目(32473007)；国家现代农业(生猪)产业技术体系(CARS-35)

Establishment of Fluorescent Microsphere Immunochromatographic Assay for Porcine Epidemic Diarrhea Virus Antigen Detection

LI Huimin^1,2, LEI Mingkai^1,2, RUAN Shengnan^1,2, LI Panpan¹, LI Wentao^1,2,3*, HE Qigai^1,2*

1. College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China;
2. National Key Laboratory of Agricultural Microbiology, Wuhan 430070, China;
3. Hubei Hongshan Laboratory, Wuhan 430070, China

Received:2024-11-27 Published:2025-09-30

摘要/Abstract

摘要： 本研究旨在建立一种快速、敏感、可定量检测猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)N蛋白抗原的荧光微球免疫层析检测方法，为猪流行性腹泻(porcine epidemic diarrhea，PED)的临床快速检测提供技术支持。本研究采用双抗体夹心法，结合荧光微球免疫层析技术，以时间分辨荧光微球(time-resolved fluorescence nanoparticles，TRFNPs)标记PEDV鼠源多克隆抗体制备荧光探针，以PEDV N蛋白单克隆抗体和羊抗鼠IgG抗体分别作检测线(T线)和质控线(C线)，组装成荧光微球免疫层析抗原检测试纸条。分别通过检测梯度稀释的培养PEDV病毒液、常见猪病病毒、模拟粪便样品和临床病料验证该方法的敏感度、特异性和临床适用性。结果显示：本方法灵敏度高，最低检测限达10^3.1 TCID₅₀·mL^-1，较商品化胶体金试纸条检测灵敏性提高至少10倍。且试纸条T/C值与病毒滴度呈现良好线性关系，可用于PEDV的定量检测。本方法操作简单、耗时短，检测时间为15 min。方法学验证显示，试纸条检测TGEV、PoRV、PDCoV、PRRSV、CSFV、ASFV时不发生交叉反应，特异性良好。试纸条的批内、批间变异系数均小于10%，重复性良好。50份临床样品的检测结果与国标RT-PCR方法总体符合率为96%，方法一致性高。本研究成功建立了基于TRFNPs的PEDV荧光微球免疫层析抗原检测方法，为临床中PEDV的快速检测提供了工具。

关键词: 猪流行性腹泻病毒, N蛋白, 时间分辨荧光微球, 免疫层析法, 定量检测

Abstract: The aim of this study is to establish a rapid, sensitive, and quantitative detection method for porcine epidemic diarrhea virus (PEDV) based on fluorescent microsphere immunochromatographic detection, providing technical support for on-site rapid detection of porcine epidemic diarrhea. By adopting a double-antibody sandwich method in conjunction with fluorescent microsphere immunochromatographic technology, the PEDV murine polyclonal antibody labeled with time-resolved fluorescent nanoparticles (TRFNPs) was prepared and utilized as a fluorescent probe. Subsequently, the anti-PEDV N protein monoclonal antibody and the goat anti-mouse IgG antibody were employed as the test line (T line) and quality control line (C line), respectively, to assemble a fluorescent microsphere immunochromatographic antigen test strip. The sensitivity of the method was evaluated by detecting a gradient dilution of PEDV. The specificity of the method was verified by testing several other common swine disease viruses. The clinical application value of the method was assessed by testing simulated fecal samples and clinical samples. The results showed that the method had high sensitivity. There was a good linear relationship between the viral titter (ranging from 10^3.3 to 10^6.0 TCID₅₀·mL^-1) and the T/C value of the test strip. The limit of detection was as low as 10^3.1 TCID₅₀·mL^-1, indicating that the method can be used for quantitative detection of PEDV. The sensitivity of the test is approximately 10 times higher than that of commercial colloidal gold test strips. The operation is simple and timesaving, with a detection time of 15 min. The method has good specificity and shows no cross-reactivity with TGEV, PoRV, PDCoV, PRRSV, CSFV, and inactivated ASFV. The results of repeatability and intermediate precision experiments showed that the intra- and inter-batch coefficients of variation of the method were both less than 10%. The method is suitable for the detection of PEDV in simulated fecal samples and clinical samples. A total of 50 clinical samples were tested simultaneously using this method and RT-PCR, the consistency rate of the test results was 96%. Here, we developed a TRFNPs-based fluorescent microsphere immunochromatographic assay (FMICA) for the detection of PEDV antigen, which might provide a tool for rapid clinical detection of PEDV.

Key words: porcine epidemic diarrhea virus (PEDV), nucleocapsid protein, time-resolved fluorescence nanoparticles (TRFNPs), fluorescent microsphere immunochromatographic assay (FMICA), quantitative detection

中图分类号:

S852.659.6

李慧敏, 雷铭楷, 阮胜男, 李盼盼, 李文涛, 何启盖. 猪流行性腹泻病毒荧光微球免疫层析抗原检测方法的建立[J]. 畜牧兽医学报, 2025, 56(9): 4572-4580.

LI Huimin, LEI Mingkai, RUAN Shengnan, LI Panpan, LI Wentao, HE Qigai. Establishment of Fluorescent Microsphere Immunochromatographic Assay for Porcine Epidemic Diarrhea Virus Antigen Detection[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4572-4580.

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猪流行性腹泻病毒荧光微球免疫层析抗原检测方法的建立

Establishment of Fluorescent Microsphere Immunochromatographic Assay for Porcine Epidemic Diarrhea Virus Antigen Detection

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