狂犬病病毒滴度直接免疫荧光检测法的建立及验证

摘要

Objective To establish and validate the direct immunofluorescence method to detect rabies virus (RV) titer.Methods The influences of inoculation method (stepwise and one-step inoculations) on titer of RV were evaluated by direct immunofluorescence assay.The optimized method was verified for inter-precision and specificity,and the results were compared with those of intracerebral titration in mice.Results The cells were inoculated by one-step inoculation.The variation coefficient of 14 test results by two personnel was only 0.04％.Specific fluorescence was observed only in RV but hot in non-RV control viruses by the method.The determination results of virus titers by direct immunofluorescence method showed positive correlation with that by intracerebral titration in mice.There is a good linear relationship between the results of the two methods (r =0.918),with linear regression equation:y =0.907x-1.566 (t =12.80,P ＜0.05).Conclusion The optimized direct immunofluorescence method is precise,specific,easy to handle and timesaving,thus can be used for the quantitative determination of RV titer.%目的 建立检测狂犬病病毒(rabies virus,RV)滴度的直接免疫荧光法,并进行方法验证.方法 采用分步接种法和一步接种法两种细胞接种方式建立检测RV滴度的直接免疫荧光法.比较不同细胞接种方式检测结果的差异,并对建立的方法进行方法学验证,考察精密度和特异性指标,用统计学方法分析数据.结果 选择一步接种法作为直接免疫荧光法的细胞接种方式;2名操作人员14次检测结果的变异系数为0.04;运用该方法检测不同病毒,仅RV组出现特异性荧光灶,而非RV对照病毒组未观察到荧光灶.采用直接免疫荧光法和小鼠脑内滴定法检测RV样品的病毒滴度,两种方法结果之间呈较好的直线相关性(r=0.918),并有正向回归关系,直线回归方程:y=0.907x-1.566(t=12.80,P＜0.05).结论 直接免疫荧光法精密度良好,特异性强,操作简便,检测周期短,可用于RV滴度的定量检测.