测定脊髓灰质炎病毒滴度的结晶紫染色法的建立及验证  

Establishment and verification of crystal violet staining method for poliovirus titer detection

作　　者：刘悦越[1] 赵岩[1] 张韵祺 赵荣荣[1] Liu Yueyue;Zhao Yan;Zhang Yunqi;Zhao Rongrong(Department of Enteric Virus Vaccine,National Institutes for Food and Drug Control,Beijing 102629,China;School of Mathematics and Statistics,Yunnan University,Kunming 650091,China)

机构地区：[1]中国食品药品检定研究院肠道病毒疫苗室,北京102629 [2]云南大学数学与统计学院,昆明650091

出　　处：《国际生物制品学杂志》2020年第2期66-71,共6页International Journal of Biologicals

基　　金：国家科技重大专项"重大新药创制"(2018ZX09739002-002)。

摘　　要：目的 建立测定脊髓灰质炎病毒(poliovirus,PV)滴度的结晶紫染色法,并对该法进行验证。方法 基于PV的半数细胞培养感染量(50% cell culture infective dose,CCID50),用结晶紫染色和酶标仪确定PV的致细胞病变效应(cytopathic effect,CPE),并验证该法的准确度、精密度、线性范围和耐用性。分别采用建立的结晶紫染色法和传统的显微镜观察法测定66批口服Ⅰ型Ⅲ型脊髓灰质炎减毒活疫苗的病毒滴度,通过Pearson相关分析评价2种检测方法的相关性。将细胞板浸泡液盲传3代,观察培养物的CPE,并通过逆转录PCR确定病毒灭活情况。结果 用建立的方法测定理论滴度8.00、6.00和4.00 lgCCID50/ml的PV样品,回收率为98%~104%。细胞板内和板间测定结果的变异系数(coefficient of variation,CV)为0%~7.85%,同一工作日和不同工作日测定结果的CV分别不超过6.44%和5.27%,不同实验人员测定结果的CV不超过5.10%。该法测定PV滴度的线性范围为2.00~8.00 lgCCID50/ml。该法具有较好的耐用性,以不同细胞浓度(F=1.624,P=0.215)或不同培养时间(F=2.731,P=0.055)培养对PV滴度的测定结果没有影响。该法与显微镜观察法的检测结果具有相关性,Pearson相关系数0.918。实验用细胞板的浸泡液在Hep-2细胞上盲传3代后,培养物未观察到CPE,逆转录PCR测定结果为阴性。结论 建立的结晶紫染色法具有较好的安全性、准确度、精密度和耐用性,适用于口服脊髓灰质炎减毒活疫苗的病毒滴度测定。Objective To establish and validate a crystal violet staining method for poliovirus(PV)titer detection.Methods Based on the 50%cell culture infective dose(CCID50)of PV,the cytopathic effect(CPE)of PV was determined by crystal violet staining and microplate reader.The accuracy,precision,linearity and robustness of the method were verified.Sixty-six lots of oral typeⅠtypeⅢpolio live vaccine were titrated by established crystal violet staining method and traditional microscope observation method,respectively.The correlation between the two methods was statistically analyzed by the Pearson’s correlation coefficient.The cell plate soaking solution was passed for 3 generations blindly,and the virus inactivation was determined by observing CPE of the culture and reverse transcription PCR(RT-PCR).Results The recovery rates of 8.00,6.00 and 4.00 lgCCID50/ml PV samples were 98%-104%using the established method.The coefficients of variation(CVs)of intra-plate and inter-plate detection results were 0%-7.85%.The CVs of intraday and interday detection results were≤6.44%and≤5.27%,respectively.The CVs of detection results from different workers were≤5.10%.The linearity range of the method was 2.00-8.00 lgCCID50/ml.The method had good robustness,and there was no effect on detection results of PV titer with different cell concentrations(F=1.624,P=0.215)or incubation time(F=2.731,P=0.055).The Pearson’s correlation coefficient 0.918 indicated that the detection results of the established method correlated with those of the microscope observation method.After the cell culture plate soaking solution was blindly passaged for 3 generations in Hep-2 cells,no CPE was observed in the culture,and RT-PCR result was negative.Conclusion The crystal violet staining method has good safety,accuracy,precision,linearity and robustness,and is suitable for the virus titer detection of oral polio live vaccine.

关 键 词：脊髓灰质炎病毒 脊髓灰质炎病毒疫苗 口服 龙胆紫 方法 

分 类 号：R3[医药卫生—基础医学]