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

Abstract

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

CLC Number:

S852.659.6

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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References 19

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病毒滴度/ (TCID₅₀·mL^-1) Virus titer	批内 Intra-assay			批间 Inter-assay
病毒滴度/ (TCID₅₀·mL^-1) Virus titer	${\bar x}$	s	CV/%	${\bar x}$	s	CV/%
10^6.0	0.453 5	0.026 4	5.82	0.4385	0.032 6	7.43
10^4.8	0.351 2	0.021 5	6.12	0.3498	0.025 7	7.35
10^3.3	0.026 1	0.002 2	8.49	0.0254	0.002 4	9.41

添加病毒/(TCID₅₀) Add viruses(TCID₅₀)	样本编号 Sample number	T/C值 T/C value	平均T/C值 Average T/C value	标准差 Standard deviation
10⁵	1-1	0.406 9	0.471 4	0.046 0
	1-2	0.510 9
	1-3	0.496 3
10⁴	2-1	0.345 2	0.351 5	0.040 8
	2-2	0.404 3
	2-3	0.305 0
10³	3-1	0.121 4	0.130 6	0.016 4
	3-2	0.153 6
	3-3	0.116 8
10²	4-1	0.018 2	0.018 8	0.002 2
	4-2	0.021 8
	4-3	0.016 4
10	5-1	0.002 4	0.003 7	0.001 1
	5-2	0.003 7
	5-3	0.005 1

方法 Method	RT-PCR
方法 Method		阳性 Positive	阴性 Negative	合计 Total
试纸条 Test strip	阳性Positive	38	0	38
	阴性Negative	2	10	12
	符合率/% Coincidence rate	95	100	96

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

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