轮状病毒减毒活疫苗感染性滴度一步法RT-qPCR检测方法的建立  

One-step real time RT-PCR in titration of rotavirus vaccine

作　　者：杜加亮[1] 刘悦越[1] 张永 赵荣荣[1] 赵一荣 韩菲 刘艳[1] DU Jialiang;LIU Yueyue;ZHANG Yong;ZHAO Rongrong;ZHAO Yirong;HAN Fei;LIU Yan(Division of Enterovirus Vaccines,National Institutes for Food and Drug Control,Beijing 102629,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

出　　处：《国际检验医学杂志》2020年第21期2570-2574,共5页International Journal of Laboratory Medicine

基　　金：十二五国家重大科技专项(2012ZX10004702);国家高技术研究发展计划(863计划)(2012AA02A402)。

摘　　要：目的建立型别特异性一步法反转录实时荧光定量聚合酶链反应检测方法,用于多价轮状病毒疫苗的鉴别和效力评价。方法针对G1、G2、G3、G4、G9型轮状病毒设计引物探针,优化反应温度和条件,使用轮状病毒疫苗株培养后收获液进行特异性、线性和范围、重复性检测,评估该反应体系的性能。结果各型别之间无交叉反应,其他肠道病毒阳性标本未见特异性扩增曲线。每个型别的稀释倍数和Ct值的线性决定系数均>95%。每个型别各浓度梯度同一检测日内和不同检测日间的变异系数均<15%,并且差异均无统计学意义(P=0.917、0.746)。结论该方法特异性、线性和范围、重复性都符合检测要求,可用于多价轮状病毒疫苗的鉴别和效力评价。Objective To establish of a serotype-specific one-step reverse transcription real-time fluorescent quantitative polymerase chain reaction for the identification and potency evaluation of multivalent rotavirus vaccines.Methods Serotype-specific pimers and probes were designed for G1,G2,G3,G4,G9 rotavirus,followed by optimization of reaction temperature and conditions.Consequently,the evaluation of specificity,linearity and range,repeatability of this method was performed by using the culture of five rotavirus vaccine strains.Results There was no cross-reaction between the five serotypes,and other enterovirus-positive samples showed no specific amplification curve.All the coefficients of determination between dilution factor and Ct value were more than 95%.The intra-and inter-day coefficients of variation were less than 15%and showed no statistic difference(P=0.917,0.746).Conclusion The specificity,linearity and range,repeatability of the method are consistent with the requirements of detection,which can be used for the identification and potency evaluation of multivalent rotavirus vaccines.

关 键 词：荧光定量聚合酶链反应 轮状病毒 疫苗 

分 类 号：R725.1[医药卫生—儿科] R44[医药卫生—临床医学]