直接免疫荧光法检测狂犬病病毒滴度条件的优化及验证

Optimization and verification of condition for determination of rabies virus titer by direct immunofluorescence assay

目的对直接免疫荧光法检测狂犬病病毒(rabies virus,RV)滴度的条件进行优化及验证。方法考察直接免疫荧光法中细胞不同接种方式(分步接种法、一步接种法)、病毒不同培养温度(34、37℃)及培养时间(6、12、24、48、72、96 h)对狂犬病病毒滴度的影响;对优化的方法进行试验间精密性和特异性验证,并与小鼠脑内滴定法的检测结果进行比较。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;34℃作为病毒培养温度;病毒培养至96 h时进行染色,判定实验结果;病毒培养至第3天时进行荧光灶计数,计算病毒液中病毒的数量。两名操作人员12次检测结果的变异系数仅为0.05%;用该方法检测不同病毒,仅狂犬病病毒组出现特异性荧光,而犬瘟热病毒和犬细小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狂犬病病毒样品的病毒滴度结果差异无统计学意义(P>0.05),具有较好的一致性。结论优化的直接免疫荧光法精密性良好,特异性强,操作简便,检测周期短,可用于狂犬病病毒滴度的定量检测。

Objective To optimize and verify the condition for determination of rabies virus titer by direct immunofluorescence assay（d FA）. Methods The influences of inoculation method（stepwise and one-step inoculations）as well as temperature（34 and 37 ℃）and time（6,12,24,48,72 and 96 h）on titer of rabies virus 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,while virus was cultured at 34 ℃ for 96 h and stained to evaluate the test result. Fluorescent foci were counted on day 3after virus culture to calculate the virus titer. The variation coefficient of 12 test results by two personnel was only 0. 05%.Specific fluorescence was observed only in rabies virus but not in canine distemper virus or canine parvovirus by the method. The determination results of virus titers by d FA showed no significant difference with that by intracerebral titration in mice（P 〉 0. 05）. Conclusion The optimized d FA is precise,specific, easy to handle and time-saving,which may be used for the quantitative determination of rabies virus titer.