基于量子点的志贺氏菌sFLISA检测方法研究

Studies on sandwich fluorescence-linked immunosorbent assay method for Shigella spp. based on quantum dots

中文关键词:  志贺氏菌  量子点  夹心荧光免疫检测

英文关键词:Shigella  quantum dots  sandwich fluorescence-linked immunosorbent assay

中文摘要:

      目的 本研究建立基于量子点的志贺氏菌sFLISA检测方法并进行验证。方法 以志贺氏菌多克隆抗体为包被抗体, 以生物素标记的志贺氏菌单克隆抗体为第二抗体, 以量子点标记的链酶亲和素进行荧光定量检测。结果 试验确定志贺氏菌sFLISA检测最佳条件: 包被抗体浓度为1.25 μg/mL, 生物素标记的单克隆抗体稀释倍数为1:400, 量子点标记的链酶亲和素稀释倍数为1:100; 特异性检验表明, sFLISA方法仅与志贺氏菌出现阳性反应, 而与大肠埃希氏菌、沙门氏菌、葡萄球菌、李斯特氏菌、变形杆菌、副溶血弧菌、芽孢杆菌等均呈阴性反应。结论 本研究建立的基于量子点的志贺氏菌sFLISA检测方法的最低检出量为3.5×104 CFU/mL, 实现了对志贺氏菌高通量定性和定量检测。

英文摘要:

      Objective To establish sFLISA (sandwich fluorescence-linked immunosorbent assay) method based on quantum dots and realize high-throughput detection of Shigella spp. Methods The study used polyclonal antibody of Shigella spp as the package antibody, and biotin-labeled clonal antibody of Shigella as the second antibody, and detected fluorescence by combination of QDs-labeled streptavidin and biotin. Results This study established sFLISA of of Shigella spp. based on quantum dots, and made validation testing using different bacteria strains. The research determined the optimum parameter of Shigella spp. in the end: the package antibody concentration was 1.25 μg/mL, biotin-labeled clonal antibody diluted multiples was 1:400, and QDs-labled streptavidin diluted multiples was 1:100. Conclusion The specificity testing showed that this method was positive only to Shigella and negative to other bacteria strains. The sensitivity of the method was 3.5×104 CFU/mL.