甲型肝炎病毒滴度检测方法的优化与验证  

Optimization and validation of titer detection method of hepatitis A virus

作　　者：杨红育 王艺博 岳立广 王拥军 裴友财 翁滨 黄晓娜 包晓婧 李莉 YANG Hong-yu;WANG Yi-bo;YUE Li-guang;WANG Yong-jun;PEI You-cai;WENG Bin;HUANG Xiao-na;BAO Xiao-jing;LI Li(Qiuility Control Department,Chungchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司质量检定室,吉林长春130012

出　　处：《微生物学免疫学进展》2021年第3期26-29,共4页Progress In Microbiology and Immunology

基　　金：吉林省科技发展计划项目(20190404001YY)。

摘　　要：目的 对优化后的甲型肝炎病毒滴度检测方法进行验证。方法 分别使用甲型肝炎病毒滴度检测的常规方法(每个细胞培养瓶中加入胰酶消化,收集后经超声破碎、氯仿抽提、聚乙二醇6000浓缩等步骤,收获病毒。滴定期间病毒在35℃吸附4h,中间轻摇2次)和优化方法(在每个细胞培养瓶中加入1%TritonX-1001mL,置35℃培养箱中30~40min,收获病毒。滴定期间病毒在35 ℃吸附1.5h,中间轻摇1次)进行滴度检测,同时对优化后的方法进行包括重复性、中间精密性(准确性)及特异性(专属性)的方法学验证,并使用SPSS 20.0统计学软件对试验数据进行统计分析。结果 不同吸附时间的病毒增殖培养实验病毒滴度3次检测结果均在8.33~8.50之间,显示优化方法与常规方法一致。优化方法的重复性验证中,取6批样品,分别检测3次,检测结果差异均无统计学意义(P1_((第1次与第2次))=0.694,P2_((第2次与第3次))=0.694,P3_((第1次与第3次))=1.000,P>0.05);中间精密性(准确性)验证中,2组实验人员分别使用常规方法和优化方法进行病毒滴度检测,其检测结果差别较小,组间差异无统计学意义(P=0.599,P>0.05);特异性(专属性)验证中,用常规检测方法及优化后的检测方法同时检测6批样品,其检测结果差异无统计学意义(P=0.203,P>0.05)。结论 甲型肝炎病毒滴度检测优化方法可以代替常规方法进行滴度检测。Objective To verify the optimized detection method of hepatitis A virus titer. Methods The titer of hepatitis A virus was detected respectively by the conventional method(the virus was collected by trypsin digestion, ultrasonic crushing, chloroform extraction, polyethylene glycol 6000 concentration and other steps. During the experiment, the virus was adsorbed at 35 ℃ for 4 h, and gently shaken twice) and the optimized method(the virus was harvest by being treated with 1 mL of 1% Triton X-100 at 35 ℃for 30 to 40 minutes. During the experiment, the virus was adsorbed at 35 ℃ for 1.5 h and gently shaken once).The repeatability, intermediate precision(accuracy) and specificity of the optimized method were verified. The test data were statistically analyzed by SPSS 20.0 statistical software. Results The virus titer data of three experiments with different adsorption time were 8.33-8.50, which showed that the virus titer detected by the optimized method was consistent with the titer detected by the conventional method. In the repeatability verification of the optimized method, six batches of samples were taken and tested for 3 times respectively, and there was not statistically significant difference(P1(the first and second time) = 0.694, P2(the second and third time) = 0.694, P3(the first and third time) = 1.000, P>0.05);in the intermediate precision(accuracy) verification, the virus titer was detected with the conventional method and the optimized method respectively by two different groups of experimenters, the difference between the two groups was not statistically significant(P=0.599, P >0.05);and in specificity verification, six batches of samples were detected simultaneously with the conventional method and the optimized method, and the difference was not statistically significant(P=0.203,P>0.05). Conclusion The optimized method can replace the conventional method for titer detection of hepatitis A virus.

关 键 词：甲肝病毒 滴度检测 方法学验证 

分 类 号：R373.21[医药卫生—病原生物学]