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。

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 immunefluotescence assy. 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 not in non-RV control viruses by the method. The determination results of virus titers by direct immunofluorescence method showed significant difference with that by intracerebral titration in mice (P<0.05), There is a good linear positive correlation coefficient between the results of the two methods (r=0.918), the 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 time-saving, which may be used for the quantitative determination of RV titer.