两种直接免疫荧光法检测狂犬病病毒滴度的验证
Validation of two direct immunofluorescence techniques for measuring rabies virus titers
于鹏程 1陶晓燕 1刘淑清 1刘茜 1张明慧 1沈立萍 1毕胜利 1朱武洋1
作者信息
- 1. 中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会生物安全重点实验室,北京 102206
- 折叠
摘要
目的 对检测狂犬病病毒(Rabies virus,RABV)滴度的荧光灶单位(Fluorescent focus unit,FFU)法和半数荧光灶数量(50％fluorescent focus infectious dose,FFD50)法两种直接免疫荧光法进行验证.方法 应用FFU法和FFD50法检测RABV不同毒株滴度,计算FFU值和FFD50值的变异系数(Coefficient of variation,CV),验证可靠性(精密度和稳定性);与标准的小鼠脑内滴定法测定的半数致死量(50％lethal dose,LD50)值进行比较,验证准确性和线性范围.结果 日间精密度和中间精密度验证显示,FFU法和FFD50法测定的CVS-11 RABV毒株滴度FFU值和FFD50值的CV均＜30％;稳定性验证显示,在改变细胞-病毒孵育时间、丙酮固定时间和染色时间后,两种方法测定的CVS-11毒株滴度FFU值和FFD50值的CV均＜30％.准确性验证显示,FFU法和FFD50法测定的CVS-11、SC16、GD1、QH2 RABV毒株滴度FFU值和FFD50值与小鼠脑内滴定法测定的LD50值均接近;病毒稀释倍数在5-1至5-7范围内,FFU和FFD50与其呈线性关系,线性回归方程分别为 y=-1.350x+7.391(R2=0.991)和 y=-1.453x+8.285(R2=0.980).结论 FFU法和FFD50法检测RABV滴度均具有良好的可靠性和准确性,为替代小鼠脑内滴定法提供了支持.
Abstract
Objective To validate two direct immunofluorescence techniques for measuring rabies virus(RABV)titers:the fluorescence focus unit(FFU)method and the 50％fluorescence focus infection dose(FFD50)method.Methods We used FFU and FFD50 to assess titers from different RABV strains,calculated coefficients of variation(C V)of FFU and FFD50 values to determine reliability(precision and stability),and compared the values with 50％lethal dose(LD50)values using the standard mouse brain titration method to validate accuracy and range of linearity.Results Repeatability and intermediate precision validation showed that CV values of FFU and FFD50 titers of the CVS-11 RABV strain were both less than 30％.Stability validation showed that after changing cell-virus incubation time,acetone fixation time,and staining time,CV values of FFU and FFD50 titers of the CVS-11 strain were all less than 30％.Accuracy validation showed that FFU and FFD50 values of CVS-11,SC16,GD1,and QH2 RABV strains were close to their LD50 values.Both FFU and FFD50 titers had linear relationships with the virus dilution ratio method ranging from 5-1 to 5-7,with linear regression equations of y=-1.350x+7.391(R2=0.991)and y=-1.453x+8.285(R2=0.980).Conclusions The FFU and FFD50 method have good reliability and accuracy for determining RABV titers,providing supportive evidence to use these method to substitute for the mouse brain titration method.
关键词
狂犬病病毒/直接免疫荧光法/荧光灶单位/半数荧光灶数量/可靠性/准确性Key words
Rabies virus/Direct fluorescent assay/Fluorescent focus unit/50％fluorescent focus infectious dose/Reliability/Accuracy引用本文复制引用
出版年
2024
NETL
NSTL
万方数据