中国兽医科学2025，Vol.55Issue(7)：905-912,8.DOI:10.16656/j.issn.1673-4696.2025.0133
胶体金免疫层析法快速定量猪流行性腹泻病毒的可行性研究
Feasibility study on rapid quantification of porcine epidemic diarrhea virus by colloidal gold immunochromatography
摘要
Abstract
A rapid and quantitative method basing on colloidal gold immunochromatography assay was established for the detection of porcine epidemic diarrhea virus(PEDV)N protein.The utility of this method in the quality control of PEDV vaccines was evaluated.The PEDV N protein was recombinantly expressed in E.coli and purified by Ni metal chelate chromatography to produce the N protein standard.The assay conditions for the colloidal gold immunochromatography were optimized,including volume,ly-sis temperature,lysis time,reaction temperature,and reaction time.A response curve between the con-centration of N protein standard and the gray level of the colloidal gold reagent test strip was estab-lished under the optimized conditions.The limit of detection,accuracy and precision were determined.Applicability of the method for detecting PEDV samples at different processing steps and for monitor-ing finished vaccines was evaluated.The correlation between the colloidal gold assay results and TCID50 assay was analyzed.The results showed that the purity of the prepared N protein standard was 94.5％.The optimal conditions for the colloidal gold immunochromatography assay were lysing the PEDV sample at room temperature for 10 min,adding 100 μL to the colloidal gold test strip,and detecting after a 20-min reaction.Under these conditions,the four-parameter fit curve showed R2=0.997 27,and the limit of de-tection was 7.14 ng/mL.The recoveries of different concentrations of N protein standard were 92.2％～111.5％,and six replicates of PEDV samples with high and low concentrations showed an RSD of less than 10％.The method was proved suitable for detecting cell culture fluid,inactivated samples and finished products of vaccines,as well as for monitoring the storage stability of vaccines.The concentration of N protein showed a strong correlation with TCID50 results,with Rs2=0.975 66 and the Pearson's correla-tion coefficient of 0.987 76.In conclusion,the colloidal gold immunochromatography assay for quanti-tative detection of PEDV N protein is fast,exhibits good linearity,accuracy and reproducibility,and is applicable for the rapid quantification of PEDV samples and the rapid assessment of vaccine quality.关键词
猪流行性腹泻病毒/胶体金免疫层析法/N蛋白/定量/疫苗Key words
porcine epidemic diarrhea virus(PEDV)/colloidal gold immunochromatography assay/N protein/quantification/vaccine分类
农业科技引用本文复制引用
宫萱,陈诗雅,杜久斌,陈晓洁,张承凤,黎明,杨延丽,贺笋..胶体金免疫层析法快速定量猪流行性腹泻病毒的可行性研究[J].中国兽医科学,2025,55(7):905-912,8.基金项目
新疆维吾尔族自治区"天池英才"青年博士项目 （）
