狂犬病病毒滴度直接免疫荧光检测法的建立及验证

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.

Establishment and validation of direct immunofluorescence method to detect rabies virus titer

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.