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

作　　者：于鹏程[1] 陶晓燕[1] 刘淑清[1] 刘茜 张明慧 沈立萍[1] 毕胜利[1] 朱武洋[1] 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)

机构地区：[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

出　　处：《中国疫苗和免疫》2024年第3期288-292,共5页Chinese Journal of Vaccines and Immunization

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.

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 

分 类 号：R331[医药卫生—人体生理学]