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

Abstract

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

CLC Number:

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.

Figures/Tables 7

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References 30

1	HUH,JUNGK,VLASOVAA N,et al.Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States[J].J Clin Microbiol,2015,53(5):1537-1548. doi: 10.1128/JCM.00031-15
2	HANF,SHANF,HOUJ,et al.Establishment and application of PDCoV antigen-specific DAS-ELISA detection method[J].BMC Vet Res,2024,20(1):342. doi: 10.1186/s12917-024-04201-w
3	KONGF Z,WANGQ H,KENNEYS P,et al.Porcine deltacoronaviruses: Origin, evolution, cross-species transmission and zoonotic potential[J].Pathogens,2022,11(1):79. doi: 10.3390/pathogens11010079
4	WANGN X,WANGZ,MAM Y,et al.Expression of codon-optimized PDCoV-RBD protein in baculovirus expression system and immunogenicity evaluation in mice[J].Int J Biol Macromol,2023,252,126113. doi: 10.1016/j.ijbiomac.2023.126113
5	ZHAOF J,LIUL T,XUM L,et al.Assessments of different inactivating reagents in formulating transmissible gastroenteritis virus vaccine[J].Virol J,2020,17(1):163. doi: 10.1186/s12985-020-01433-8
6	NIEDERWERDERM C,HESSER A.Swine enteric coronavirus disease: A review of 4 years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the United States and Canada[J].Transbound Emerg Dis,2018,65(3):660-675. doi: 10.1111/tbed.12823
7	常新见,周金柱,殷杰,等.2017-2019年华东地区猪场主要病毒性腹泻病原调查[J].畜牧兽医学报,2020,51(12):3141-3150. doi: 10.11843/j.issn.0366-6964.2020.12.023
	CHANGX J,ZHOUJ Z,YINJ,et al.Investigation on pathogens of major viral diarrhea in pig farms in east china from 2017 to 2019[J].Acta Veterinaria et Zootechnica Sinica,2020,51(12):3141-3150. doi: 10.11843/j.issn.0366-6964.2020.12.023
8	WANGZ,QUK,LIJ H,et al., Prevalence and potential risk factors of PDCoV in pigs based on publications during 2015-2021 in China: Comprehensive literature review and meta-analysis[J].Microb Pathog,2023,179,106118. doi: 10.1016/j.micpath.2023.106118
9	WANGS,LIW J,WANGZ S,et al.Emerging and reemerging infectious diseases: global trends and new strategies for their prevention and control[J].Sig Transduct Target Ther,2024,9(1):223. doi: 10.1038/s41392-024-01917-x
10	OKDAF S,LAWSONX,LIUA,et al.Development of monoclonal antibodies and serological assays including indirect ELISA and fluorescent microsphere immunoassays for diagnosis of porcine deltacoronavirus[J].BMC Vet Res,2016,12,95. doi: 10.1186/s12917-016-0716-6
11	WANGW L,ZHANGY N,YANGH C.Development of a nucleocapsid protein-based blocking ELISA for the detection of porcine deltacoronavirus antibodies[J].Viruses,2022,14(8):1815. doi: 10.3390/v14081815
12	路浩,丁雪燕,袁晋,等.猪肠道冠状病毒检测方法的研究进展[J].中国兽医科学,2024,54(10):1391-1398.
	LUH,DINGX Y,YUANJ,et al.Research progress on detection methods of swine enteric coronavirus[J].Chinese Veterinary Science,2024,54(10):1391-1398.
13	LABORIEE,LE-MINHV,MAIT D,et al.Analytical methods of antibody surface coverage and orientation on bio-functionalized magnetic beads: application to immunocapture of TNF-α[J].Anal Bioanal Chem,2021,413(25):6425-6434. doi: 10.1007/s00216-021-03608-w
14	VINDUSKAV,GALLOPSC E,O'CONNORR,et al.Exosomal surface protein detection with quantum dots and immunomagnetic capture for cancer detection[J].Nanomaterials (Basel),2021,11(7):1853. doi: 10.3390/nano11071853
15	黄婧洁,李苗,陈莹娴,等.基于免疫磁珠净化间接竞争酶联免疫吸附试验检测氟喹诺酮类药物[J].畜牧兽医学报,2023,54(2):766-778. doi: 10.11843/j.issn.0366-6964.2023.02.033
	HUANGJ J,LIM,CHENY X,et al.Indirect competitive enzyme-linked immunosorbent assay for the detection of fluorquinolones based on immunomagnetic bead purification[J].Acta Veterinaria et Zootechnica Sinica,2023,54(2):766-778. doi: 10.11843/j.issn.0366-6964.2023.02.033
16	XUT,ZHOUY C,LIUZ Y,et al.Prevalence and genetic diversity of porcine epidemic diarrhea virus in Southwest China during 2020-2022[J].Sci Rep,2024,14(1):29124. doi: 10.1038/s41598-024-80844-x
17	ZHANGF F,LUOY Y,LINC,et al.Epidemiological monitoring and genetic variation analysis of pathogens associated with porcine viral diarrhea in southern China from 2021 to 2023[J].Front Microbiol,2024,15,1303915. doi: 10.3389/fmicb.2024.1303915
18	王挺,李风云,葛生虎,等.2020年~2021年河北省4种猪腹泻病毒流行情况调查及PEDV分离株S1和ORF3基因序列的遗传变异分析[J].中国预防兽医学报,2023,45(5):473-479.
	WANGT,LIF Y,GES H,et al.Epidemic survey of four diarrhea viruses in pig farms and genetic variation analysis of S1 and ORF3 gene sequences of PEDV strains in Hebei Province from 2020 to 2021[J].Chinese Journal of Preventive Veterinary Medicine,2023,45(5):473-479.
19	韦学雷,梁青青,曹贝贝,等.猪Delta冠状病毒、猪传染性胃肠炎病毒和猪流行性腹泻病毒多重RT-PCR检测方法的建立及应用[J].中国兽医学报,2018,38(1):11-16.
	WEIX L,LIANGQ Q,CAOB B,et al.Establishment and application of a multiplex RT-PCR assay for simultaneous detection of porcine delta coronavirus, porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus[J].Chinese Journal of Veterinary Medicine,2018,38(1):11-16.
20	辛忠昊,焦安琪,朱彤,等.四种常见猪肠道病毒多重RT-PCR检测方法的建立及临床应用[J].微生物学通报,2022,49(12):5126-5137.
	XINZ H,JIAOA Q,ZHUT,et al.Establishment and clinical application of multiplex RT-PCR assay for four common porcine enteroviruses[J].Microbiological Bulletin,2022,49(12):5126-5137.
21	闫晓光,袁晋,胡文阳,等.猪冠状病毒通用荧光定量PCR检测方法的建立[J].中国兽医学报,2023,43(1):16-22.
	YANX G,YUANJ,HUW Y,et al.Establishment of universal fluorescence quantitative PCR detection method for four common porcine enteroviruses[J].Chinese Journal of Veterinary Medicine,2023,43(1):16-22.
22	LAZOVC M,PAPETTIA,BELSHAMG J,et al.Multiplex real-time RT-PCR assays for detection and differentiation of porcine enteric coronaviruses[J].Pathogens,2023,12(8):1040. doi: 10.3390/pathogens12081040
23	张利卫,曹贝贝,李炳晓,等.新发猪Delta冠状病毒和猪流行性腹泻病毒SYBR Green Ⅰ双重荧光RT-PCR检测方法的建立及应用[J].中国兽医学报,2018,38(4):618-624.
	ZHANGL W,CAOB B,LIB X,et al.Establishment and application of a duplex SYBR Green Ⅰ real-time RT-PCR assay for simultaneous detection of emerging PDCoV and PEDV[J].Chinese Journal of Veterinary Medicine,2018,38(4):618-624.
24	舒佳新,陈传君,谢礼,等.重组酶聚合酶扩增技术快速检测猪肉及其制品中ASFV、PDCoV和SVA[J].中国食品卫生杂志,2023,35(7):1013-1020.
	SHUJ X,CHENC J,XIEL,et al.Rapid detection of ASFV, PDCoV, and SVA in pork and its products by recombinant enzyme polymerase amplification[J].Chinese Journal of Food Hygiene,2023,35(7):1013-1020.
25	CONGX,ZHUY J,LIUX C,et al.Establishment of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of swine acute diarrhea syndrome coronavirus (SADS-CoV)[J].BMC Vet Res,2022,18(1):369. doi: 10.1186/s12917-022-03465-4
26	EL-THOLOTHM,BAIH,MAUKM G,et al.A portable, 3D printed, microfluidic device for multiplexed, real time, molecular detection of the porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine deltacoronavirus at the point of need[J].Lab Chip,2021,21(6):1118-1130. doi: 10.1039/D0LC01229G
27	韩郁茹,石达,张记宇,等.猪急性腹泻综合征冠状病毒RT-LAMP快速检测方法的建立与应用[J].中国预防兽医学报,2021,43(1):35-39.
	HANY R,SHID,ZHANGJ Y,et al.Development and application of RT-LAMP method for rapid detection of SADS-CoV[J].Chinese Journal of Preventive Veterinary Medicine,2021,43(1):35-39.
28	臧冉,徐飞飞,郑丹阳,等.PEDV与TGEV双重荧光RT-LAMP检测方法的建立与评价[J].中国兽医学报,2024,44(8):1600-1610.
	ZANGR,XUF F,ZHENGD Y,et al.Establishment and evaluation of a dual fluorescent RT-LAMP assay for PEDV and TGEV detection[J].Chinese Journal of Veterinary Medicine,2024,44(08):1600-1610.
29	ZHANGY X,SONGY,RENH J,et al.Preparation of a single-chain antibody against nucleocapsid protein of porcine deltacoronavirus by phage display technology[J].Viruses,2022,14(4):772. doi: 10.3390/v14040772
30	CHENY W,ZHANGY Z,WANGX,et al.Transmissible gastroenteritis virus: An update review and perspective[J].Viruses,2023,15(2):359. doi: 10.3390/v15020359

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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