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目的应用细胞荧光转化灶(fluorescence focus units assay,FFU)法检测狂犬病病毒滴度。方法将狂犬病病毒稀释后接种BSR细胞,培养22~24 h,用FITC标记的抗狂犬病病毒特异性抗体进行染色,计数病毒感染细胞的荧光灶数,计算病毒滴度。采用乳鼠半数致死量(median lethal dose,LD50)法和建立的方法分别测定14批狂犬病病毒滴度,分析两者间的相关性;取同一份病毒液,于不同时间点检测3次,每个时间点重复检测6次,验证方法的重复性;取3份滴度不同的病毒液,由2个操作者在不同时间重复测定3次,验证方法的中间精密性;将同一份病毒液按2倍梯度稀释,共9个稀释度,分别检测病毒滴度,重复测定3次,确定该方法的线性范围。应用建立的方法检测38批CTNCEC株狂犬病病毒滴度。结果乳鼠LD50法测定14批狂犬病病毒的-Lg LD50值范围为4.20~8.19,FFU法Lg FFU值范围为3.76~7.69,两种方法检测结果趋势相同,线性回归相关系数R-Sq(调整)=96.1%,存在良好的正相关性;同一个时间点检测6次及不同时间点检测3次的CV值均小于2.00%;两个操作者在不同时间重复测定3次的CV值均小于8.00%,两个操作者之间的检测结果差异无统计学意义(P>0.05);样品浓度与Lg FFU呈良好的线性关系,R-Sq(调整)=99.3%,线性范围为2.26~6.12。38批CTNCEC株狂犬病病毒滴度平均Lg FFU在3.63~7.69之间,SD值在0.021~0.57之间,CV值小于10.00%。结论建立的细胞FFU法准确性、重复性、中间精密度好,该方法可用于检测狂犬病病毒滴度。
Objective To detect rabies virus titer by fluorescence focus units assay (FFU). Methods BSR cells were inoculated diluted with rabies virus and cultured for 22-24 h. The cells were stained with FITC-labeled anti-rabies virus-specific antibody and the number of virus-infected foci was counted to calculate the virus titer. 14 rabies virus titers were determined by the median lethal dose (LD50) method and the established method, respectively. The correlation between them was analyzed. The same virus solution was used to detect the rabies virus at different time points for 3 times. A time point of repeated testing 6 times to verify the repeatability of the method; take 3 different titer of virus solution, repeated by two operators at different times 3 times to verify the accuracy of the method; the same virus liquid press 2 times gradient dilution, a total of 9 dilutions were detected virus titer, repeated determination of 3 times to determine the linear range of the method. 38 batches of CTNCEC strain of rabies virus were tested by established method. 14 measurement results of rabies virus batch method -Lg suckling mice LD50 LD50 values ranging from 4.20 ~ 8.19, Lg FFU FFU value in the range of 3.76 ~ 7.69 France, the same trend of these two detection methods, the linear regression correlation coefficient R-Sq (adjusted) = 96.1%, there is a good positive correlation; CV values of 6 times at the same time point and 3 times at different time points are all less than 2.00%; CV values of 3 times repeated by two operators at different time are less than 8.00 %, no difference between the two detection results of the operator significant (P> 0.05); and Lg FFU sample concentration showed a good linear relationship, R-Sq (adjusted) = 99.3%, the linear range of 2.26 to 6.12. The average titer of rabies virus of 38 batches of CTNCEC strains was between 3.63 and 7.69, the SD value was between 0.021 and 0.57, and the CV value was less than 10.00%. Conclusion The established cell FFU method is accurate, reproducible and has good intermediate precision. This method can be used to detect rabies virus titer.