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为建立猪传染性胃肠炎病毒(TGEV)直观、特异和快速的检测方法,将TGEV接种PK-15细胞后,以抗TGEV N蛋白的单克隆抗体为一抗,荧光素FITC标记的山羊抗小鼠Ig G为二抗,建立了TGEV的间接免疫荧光(IFA)检测方法。结果显示,TGEV的最佳接种效价为1×102TCID50,培养时间为24 h,细胞最佳固定液为40 m L/L多聚甲醛,最佳封闭条件为10 g/L BSA 37℃封闭30 min,一抗的最佳使用浓度为1 ng/L,二抗的最佳稀释度为1∶100。对常见的猪流行性腹泻病毒、猪轮状病毒和猪圆环病毒2型等猪病病原检测均为阴性。结果表明,建立的检测方法对TGEV具有良好的特异性。本研究建立的方法为TGEV的实验室诊断及TGEV在感染细胞中的定位和动态分布提供了有效的检测手段。
In order to establish an intuitionistic, specific and rapid detection of porcine transmissible gastroenteritis virus (TGEV), TGEV was inoculated on PK-15 cells and the monoclonal antibody against TGEV N protein was used as primary antibody. FITC-labeled goat anti-fluorescein Mouse Ig G is a secondary antibody, established indirect immunofluorescence (IFA) detection of TGEV. The results showed that the best inoculation titer of TGEV was 1 × 102TCID50, the culture time was 24 h, and the optimal cell fixing solution was 40 m L / L paraformaldehyde. The optimal blocking conditions were 10 g / L BSA, 37 ℃ closed 30 min, the optimal concentration of primary antibody is 1 ng / L and the optimal dilution of secondary antibody is 1: 100. The common pig epidemic diarrhea virus, porcine rotavirus and porcine circovirus type 2 and other swine pathogens were negative. The results show that the established detection method has good specificity for TGEV. The method established in this study provided an effective method for the laboratory diagnosis of TGEV and the localization and dynamic distribution of TGEV in infected cells.