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目的建立牛血清中牛病毒直接免疫荧光检测方法,并进行验证。方法建立直接免疫荧光法检测牛血清中牛病毒,对细胞接种浓度和血清浓度、病毒接种量、荧光抗体浓度、染色温度及时间进行优化;对优化的方法进行重复性、特异性、灵敏度验证,并与细胞培养法的检测结果进行比较。结果优化的试验条件为:Vero和BT细胞的接种浓度分别为0.5×105和1.0×105个/ml,血清浓度为5%,病毒接种量为100~300 CCID50,荧光抗体1∶10稀释,4℃染色12 h以上,但不超过24 h。2名实验人员按建立的方对3批牛血清分别进行3次检测,均未检出6种牛病毒;每种病毒仅在相应抗体进行染色时出现荧光;该方法检测6种牛病毒的灵敏度较高。分别采用细胞培养法和直接免疫荧光法检测15批新生牛血清和5批胎牛血清,结果均未检出牛病毒。结论建立的牛血清中牛病毒直接免疫荧光检测方法重复性好,特异性强,灵敏度高,操作简便,与细胞培养法的检测结果无差异,可应用于牛血清中牛病毒的检测。
Objective To establish a method for the direct immunofluorescence detection of bovine virus in bovine serum and to verify it. Methods Direct immunofluorescence assay was used to detect bovine virus in bovine serum. The inoculum concentration, serum concentration, virus inoculum size, concentration of fluorescent antibody, dyeing temperature and time were optimized. The optimized method was used to verify the repeatability, specificity and sensitivity, And compared with the cell culture test results. Results The optimized experimental conditions were as follows: the inoculum concentrations of Vero and BT cells were 0.5 × 105 and 1.0 × 105 cells / ml respectively, the serum concentration was 5%, the virus inoculum size was 100-300 CCID50, 1:10 dilution of fluorescent antibody and 4 ℃ staining more than 12 h, but not more than 24 h. Two experimentally-tested three batches of bovine serum were tested according to the established protocol, respectively. None of the six bovine viruses was detected. Each virus showed fluorescence only when the corresponding antibodies were stained. The method was used to detect the sensitivity of six bovine viruses Higher. Fifteen batches of newborn calf serum and five batches of fetal calf serum were detected by cell culture and direct immunofluorescence, respectively. No bovine virus was detected. Conclusion The method for direct immunofluorescence detection of bovine serum in bovine serum with good reproducibility, specificity, high sensitivity, simple operation and no difference with the cell culture method can be applied to the detection of bovine virus in bovine serum.