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。
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(FFD_(50))method.Methods We used FFU and FFD_(50)to assess titers from different RABV strains,calculated coefficients of variation(CV)of FFU and FFD_(50)values to determine reliability(precision and stability),and compared the values with 50%lethal dose(LD_(50))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 FFD_(50)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 FFD_(50)titers of the CVS-11 strain were all less than 30%.Accuracy validation showed that FFU and FFD_(50)values of CVS-11,SC16,GD1,and QH2 RABV strains were close to their LD_(50)values.Both FFU and FFD_(50)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(R~2=0.991)and y=-1.453x+8.285(R~2=0.980).Conclusions The FFU and FFD_(50)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.
作者
于鹏程
陶晓燕
刘淑清
刘茜
张明慧
沈立萍
毕胜利
朱武洋
Yu Pengcheng;Tao Xiaoyan;Liu Shuqing;Liu Qian;Zhang Minghui;Shen Liping;Bi Shengli;Zhu Wuyang(National Health Commission Key Laboratory of Biosafety,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)
出处
《中国疫苗和免疫》
CSCD
北大核心
2024年第3期288-292,共5页
Chinese Journal of Vaccines and Immunization
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
Rabies virus
Direct fluorescent assay
Fluorescent focus unit
50%fluorescent focus infectious dose
Reliability
Accuracy
