O、A、Asia 1型口蹄疫病毒胶体金定量检测免疫层析方法的建立

doi:10.11843/j.issn.0366-6964.2021.04.019

摘要/Abstract

摘要： 旨在建立口蹄疫病毒（foot-and-mouth disease virus，FMDV）抗原血清型的快速分型和定量的检测方法，利用双抗体夹心法，将口蹄疫病毒的兔抗及豚鼠抗体作为标记胶体金与NC膜检测带的原料，分别制备出检测O、A、Asia 1血清型的3种层析试纸卡。通过对标定的抗原标准品146S检测，拟合出定量标准曲线。免疫层析方法的质量验证通过特异性、敏感性、重复性及与蔗糖密度梯度法（sucrose density gradient，SDG）的相关性进行评价。结果显示：建立的快速定量检测方法，3种血清型口蹄疫病毒间无交叉反应，同时与其他非口蹄疫病毒，如塞内卡病毒A型（SVA）、猪水疱病病毒（SVDV）、猪水疱性口炎病毒（VSV）无非特异反应；敏感性研究，对O、A、Asia 1型病毒的最低检出量分别为0.567、0.693、0.219 μg·mL^-1，拟合的3条标准曲线的线性相关系数R²>0.97，新建立方法与蔗糖密度梯度法检测结果的相关系数均>0.9，3种层析试纸卡的变异系数均小于10%。综上表明，建立的口蹄疫O、A、Asia1型病毒胶体金免疫层析定量检测试纸卡方法，可以用于口蹄疫病毒抗原快速鉴别、血清分型与146S定量检测。

关键词: 口蹄疫病毒, 胶体金免疫层析, 定量检测, 146S

Abstract: To establish a rapid genotyping and quantitative detection method for the foot-and-mouth disease virus (FMDV), the rabbit anti-FMDV and guinea pig anti-FMDV antibodies were used for labeling colloidal gold and NC membrane with strips by double antibody sandwich method. And three types chromatographic strips for detecting serotype O, A and Asia 1 were prepared. A quantitative standard curve was fitted by detecting the 146S of the calibrated reference materials. The quality verification of the immunochromatographic method was evaluated by specificity, sensitivity，reproducibility and correlation with sucrose density gradient. The quantitative detection method established in this study was distinctive, and there was no cross-reactivity among three serotypes of FMDV. This method had no cross reaction with Senecavirus A(SVA), swine vesicular disease virus (SVDV) and vesicular stomatitis virus (VSV). The limits of quantitative detection of the serotype O, A and Asia 1 viruses were 0.567, 0.693 and 0.219 μg·mL^-1 respectively. The assay also showed good linearity with a linear fitting coefficient of determination (R²) higher than 0.97. The correlation coefficient between the newly established method with 3 types lateral flow assay and sucrose density gradient was higher than 0.9. The variation coefficients of the three kinds of lateral flow assay are less than 10%.The results indicated that colloidal gold immunochromatographic quantitative assays for serotype O, A and Asia 1 FMDV could be used for identification of antigen, serotyping and quantitative detection of 146S.

Key words: foot-and-mouth disease virus, colloidal gold immunochromatographic assay, quantitative detection, 146S

中图分类号:

S854.43

李昕, 孙燕燕, 林密, 陈夏辉, 李峰松, 包艳芳, 杨光, 蒋韬. O、A、Asia 1型口蹄疫病毒胶体金定量检测免疫层析方法的建立[J]. 畜牧兽医学报, 2021, 52(4): 1042-1052.

LI Xin, SUN Yanyan, LIN Mi, CHEN Xiahui, LI Fengsong, BAO Yanfang, YANG Guang, JIANG Tao. Establishment of Colloidal Gold Immunochromatographic Assay for Quantitative Detection of Foot-and-mouth Disease Virus Serotype O, A and Asia 1[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(4): 1042-1052.

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