诺如病毒常见流行株胶体金免疫层析快速检测方法

Colloidal gold immunochromatographic assay for rapid detection of Norovirus epidemic strains

摘要:

【背景】诺如病毒是全球引发急性胃肠炎的最主要病原之一，具有丰富的遗传多样性。【目的】建立一种简便快捷、适用于诺如病毒常见流行株的胶体金免疫层析检测方法。【方法】将抗诺如病毒衣壳蛋白的单克隆抗体1B10作为金标抗体、抗诺如病毒衣壳蛋白的单克隆抗体1D6作为检测线、羊抗鼠抗体作为质控线组装成胶体金试纸条。对试纸条的组装条件进行优化，确定最佳标记pH、金标抗体最佳浓缩比例及检测线(test line，T线)、质控线(control line，C线)最佳划线浓度等。对新方法进行性能评价，包括灵敏性试验、特异性试验、保存期试验及符合率实验等。【结果】所建立的诺如病毒胶体金试纸条检测方法最低检测浓度为5.9×105 copies/μL。本方法与常见的腹泻病毒，如轮状病毒、星状病毒、腺病毒、肠道病毒均无交叉反应。批次间与批次内重复较好，保存期试验表明试纸条至少可以室温密封干燥保存一年时间。应用所建立的胶体金检测方法对24份临床粪便样本进行检测，检测结果与实时荧光RT-PCR方法的阳性符合率约为83% (15/18)，常见流行株GII.2型、GII.4型、GII.17型均被成功检出。【结论】建立的胶体金试纸条检测方法具有较好的特异性与稳定性，可用于诺如病毒常见流行株检测及大规模流行病学调查。

关键词:诺如病毒，胶体金，试纸条，流行株，免疫检测

Abstract:

[Background] Norovirus is one of the most important pathogens causing acute gastroenteritis, and it is rich in genetic diversity. [Objective] This study intends to establish a simple and rapid method for colloidal gold immunochromatographic assay of Norovirus epidemic strains. [Methods] Monoclonal antibody 1B10 against Norovirus capsid protein labeled with colloidal gold. Monoclonal antibody 1D6 against Norovirus capsid protein and goat anti-mouse antibody was blotted on nitrocellulose membrane as test line and control line, respectively. Optimize the assembly conditions of the test strip, including the labeling pH, the concentration of the gold-labeled antibody and the concentration of the test line and control line. Evaluate the performance of the method, including sensitivity test, specificity test, shelf life test and coincidence rate. Finally, the method was applied to the detection of clinical samples to evaluate its application effect. [Results] The limit of detection was 5.9×105 copies/μL. This method did not cross-react with common diarrhea viruses such as Rotavirus, Astrovirus, Adenovirus, and Enterovirus. This method has a good repeatability. The test strip can be stored for at least one year at room temperature. A total of 24 samples were detected by the method. The positive coincidence rate between the test strip and the real-time fluorescence quantitative RT-PCR method was about 83% (15/18). Epidemic strains GII.2, GII.3, GII.4, and GII.17 were successfully detected by the method. [Conclusion] The established colloidal gold test strip method has good specificity and stability, and can be used for the detection of Norovirus epidemic strains and large-scale epidemiological investigation.

Key words:Norovirus, Colloidal gold, Test strip, Epidemic strains, Immunoassay