基于免疫磁珠的智能手机辅助比色传感平台用于PDCoV/TGEV IgG抗体的快速检测

doi:10.11843/j.issn.0366-6964.2025.09.034

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (9): 4559-4571.doi: 10.11843/j.issn.0366-6964.2025.09.034

基于免疫磁珠的智能手机辅助比色传感平台用于PDCoV/TGEV IgG抗体的快速检测

刘君君¹(), 郭东辉¹^,²^,³(), 刘缓缓¹, 宋润泽¹, 赵赛娅¹, 杨钧尧¹, 魏战勇², 项玉强¹^,²^,³^,*(), 陈丽颖¹^,²^,³^,*()

1. 河南农业大学动物医学院, 郑州 450046
2. 动物病原与生物安全教育部重点实验室, 郑州 450046
3. 河南省动物性食品病原体监控重点实验室, 郑州 450046

收稿日期:2024-12-17 出版日期:2025-09-23 发布日期:2025-09-30
通讯作者: 项玉强,陈丽颖 E-mail:ljj_box@163.com;guodonghui0808@126.com;yuqiangxiang@henau.edu.cn;chliying@henau.edu.cn
作者简介:刘君君(1995-)，女，河南开封人，硕士，主要从事动物疫病快速诊断方法的研究，E-mail: ljj_box@163.com
郭东辉(1986-)，男，河南新乡人，博士生，主要从事动物传染病学研究，E-mail: guodonghui0808@126.com
第一联系人：
刘君君和郭东辉为同等贡献作者
基金资助:
国家重点研发计划项目(2024YFD1800405；2023YFD1801303)；国家自然科学基金青年基金项目(32302891)；河南省重点研发专项项目(231111113100)；河南省自然科学基金交叉学科创新研究群体项目(232300421001)；河南农业大学“拔尖人才”项目(30501279)

Rapid Visual Detection for PDCoV/TGEV IgG Antibodies Using Smartphone-Assisted Colorimetric Sensing Platform based on Immunomagnetic Beads

LIU Junjun¹(), GUO Donghui¹^,²^,³(), LIU Huanhuan¹, SONG Runze¹, ZHAO Saiya¹, YANG Junyao¹, WEI Zhanyong², XIANG Yuqiang¹^,²^,³^,*(), CHEN Liying¹^,²^,³^,*()

1. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China
2. Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, Zhengzhou 450046, China
3. Henan Province Key Laboratory for Animal Food Pathogens Surveillance, Zhengzhou 450046, China

Received:2024-12-17 Online:2025-09-23 Published:2025-09-30
Contact: XIANG Yuqiang, CHEN Liying E-mail:ljj_box@163.com;guodonghui0808@126.com;yuqiangxiang@henau.edu.cn;chliying@henau.edu.cn

摘要/Abstract

摘要：

本研究旨在建立灵敏、经济、便携和快速现场可视化检测猪丁型冠状病毒(PDCoV)和猪传染性胃肠炎病毒(TGEV)IgG抗体的方法。本研究分别将PDCoV和TGEV的N蛋白偶联到羧基磁珠表面形成“PDCoV-N/TGEV-N蛋白特异性IgG-酶标记抗体”三聚体复合物检测体系，建立基于免疫磁珠的间接ELISA方法，结合智能手机辅助比色传感平台，用于猪血清中PDCoV和TGEV IgG抗体的快速可视化检测。结果显示：以检测PDCoV IgG抗体为例，优化了免疫磁珠间接ELISA的反应条件，其最佳反应条件：磁珠用量25 μg、抗原包被浓度5 μg·mL^-1(30 min)、封闭液为2.5% BSA+脱脂奶粉混合液(37 ℃封闭2 h)、血清孵育60 min、酶标二抗1∶8 000稀释(孵育30 min)、TMB显色10 min。在该最佳反应条件下，该方法对PDCoV和TGEV抗体检测效价分别达1∶32 768和1∶8 192，批内/批间变异系数均 < 8%，且与7种常见猪源病原体阳性血清无交叉反应。临床验证显示，与中和试验相比，80份样本的检测总符合率达95%(PDCoV)和96.25%(TGEV)。本研究建立的基于免疫磁珠的智能手机辅助比色传感平台的检测方法具有较高的特异性和敏感性，且重复性好，可用于PDCoV和TGEV临床血清样本的现场快速可视化检测，将为PDCoV/TGEV的流行病学调查和疫苗免疫效果评价提供可靠工具。

关键词: 免疫磁珠, 猪丁型冠状病毒, 猪传染性胃肠炎病毒, N蛋白, 智能手机, 比色检测

Abstract:

The goal of this study was to develop a sensitive, cost-effective, portable, rapid visual detection method for porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) IgG antibodies. This study conjugated the N proteins of PDCoV and TGEV to carboxylated magnetic bead surfaces to form a trimeric complex detection system of "PDCoV-N/TGEV-N protein-specific IgG-enzyme-labeled antibody". We established an immunomagnetic bead-based indirect ELISA method combined with a smartphone-assisted colorimetric sensing platform for rapid visual detection of PDCoV and TGEV IgG antibodies in porcine serum. Optimized the experimental conditions of IMB-based indirect ELISA for the detection of PDCoV IgG antibody, with the optimal reaction conditions being as follows: magnetic beads dosage 25 μg; antigen coating concentration 5 μg·mL^-1 (30 min); blocking solution containing 2.5% BSA combined with skim milk powder (37℃ for 2 h); serum incubation for 60 min; enzyme-labeled secondary antibody at 1∶8 000 dilution (30 min); TMB substrate development for 10 min. Under these optimized conditions, this method demonstrated antibody detection titers of 1∶32 768 for PDCoV and 1∶8 192 for TGEV, with intra-/inter-assay coefficients of variation both < 8%, and showed no cross-reactivity with positive sera against seven common porcine pathogens. Clinical validation revealed overall agreement rates of 95% (PDCoV) and 96.25% (TGEV) compared with neutralization assays in 80 clinical samples. The detection method established in this study, based on an immunomagnetic bead-integrated smartphone-assisted colorimetric sensing platform, exhibits high specificity, sensitivity, and reproducibility. This field-deployable approach enables rapid visual on-site detection of PDCoV and TGEV in clinical serum samples, offering a robust solution for epidemiological monitoring and vaccine effectiveness assessment in PDCoV/TGEV control programs.

Key words: immunomagnetic beads, porcine deltacoronavirus, transmissible gastroenteritis virus, N protein, Smartphone, colorimetric detection

中图分类号:

刘君君, 郭东辉, 刘缓缓, 宋润泽, 赵赛娅, 杨钧尧, 魏战勇, 项玉强, 陈丽颖. 基于免疫磁珠的智能手机辅助比色传感平台用于PDCoV/TGEV IgG抗体的快速检测[J]. 畜牧兽医学报, 2025, 56(9): 4559-4571.

LIU Junjun, GUO Donghui, LIU Huanhuan, SONG Runze, ZHAO Saiya, YANG Junyao, WEI Zhanyong, XIANG Yuqiang, CHEN Liying. Rapid Visual Detection for PDCoV/TGEV IgG Antibodies Using Smartphone-Assisted Colorimetric Sensing Platform based on Immunomagnetic Beads[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4559-4571.

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基于免疫磁珠的智能手机辅助比色传感平台用于PDCoV/TGEV IgG抗体的快速检测

Rapid Visual Detection for PDCoV/TGEV IgG Antibodies Using Smartphone-Assisted Colorimetric Sensing Platform based on Immunomagnetic Beads

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血清中和试验 Serum neutralization test		酶标仪检测结果(OD_{450 nm}) Microplate reader test result (OD_{450 nm})			智能手机检测结果(RGB) Smartphone test result (RGB)
血清中和试验 Serum neutralization test		阳性 Positive	阴性 Negative	符合率/% Coincidence rate	阳性 Positive	阴性 Negative	符合率/% Coincidence rate
阳性血清 Positive sera	50	46	4	92	46	4	92
阴性血清 Negative sera	30	0	30	100	0	30	100
合计 Total	80	46	34	95	46	34	95