荧光MAPREC分析Ⅰ型Sabin株脊髓灰质炎病毒突变率方法的建立和应用  被引量:1

Set up and application of fluorescent MAPREC in quantitation of mutant rate in type Ⅰ Sabin polio vaccine strain

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作  者:徐康维[1] 张平 英志芳[1] 魏文明 何蕊 王剑锋[1] 赵慧[1] 李长贵[1] XU Kang-wei;ZHANG Ping;YING Zhi-fang;WEI Wen-ming;HE Rui;WANG Jian-feng;ZHAO Hui;LI Chang-gui(National Institutes for Food and Drug Control Division of Respiratory Virus Vaccines, Beijing, 100050, China)

机构地区:[1]中国食品药品检定研究院呼吸道病毒疫苗室,北京100050 [2]国家食品药品监督管理总局食品药品审核查验中心药品化妆品核查处,北京100061 [3]北京天坛生物制品有限公司质量保证部,北京100176

出  处:《微生物学免疫学进展》2017年第6期56-60,共5页Progress In Microbiology and Immunology

基  金:国家国际科技合作专项(2013DFA31680);国家科技重大专项(2015ZX09101044)

摘  要:目的使用CY5荧光标记引物建立定量检测I型Sabin株脊髓灰质炎病毒480-A和525-C突变率的MAPREC。方法提取I型Sabin株脊髓灰质炎病毒样品RNA,反转录合成c DNA后,进行非对称PCR扩增以生成单链DNA,之后采用CY5荧光标记引物进行一步扩增反应以合成双链产物,以DNA内切酶Dde I和Nci I酶切后电泳分离,获取荧光信号,计算样品的突变率。检测4个国际标准品(100%DNA突变标准品、IS DNA标准品、LMVR标准品和HMVR标准品)的突变率,重复5次,验证方法的准确性和精密度,并进行初步应用。结果使用CY5荧光标记引物,建立了定量检测I型Sabin株脊髓灰质炎病毒480-A和525-C突变率的MAPREC。100%DNA突变标准品、IS DNA标准品、LMVR标准品以及HMVR标准品的标示值分别为100.00%、2.00%、1.84%、2.56%,检测结果分别为95.09%、2.06%、1.45%、1.92%,各标准品的实验间CV值均<15%。I型Sabin株口服脊髓灰质炎减毒活疫苗工作种子、单次收获液和原液的突变率为1.05%~1.22%,批间一致性良好。结论初步建立了基于荧光素标记的MAPREC,可以代替同位素法进行I型Sabin株脊髓灰质炎病毒突变率的定量检测。To set up a CY5 fluorescent MAPREC in quantitation of mutant rate in type I Sabin polio vaccine strain instead of radiolabelling.Methods Type I Sabin polio virus RNA was isolated for c DNA synthesis,followed by unsymmetrical PCR to generate a single-stranded DNA.A fluorescently labelled primer was subsequently used to prime a second-strand DNA synthesis by a one-step DNA polymerase reaction.Labelled double stranded products were then digested with the specific restriction enzyme Dde I and Nci I then followed by separation in electrophoresis.The mutant contents of the samples were then calculated.Four international standard materials(100% DNA,IS DNA,LMVR and HMVR) were tested 5 times to evaluate accuracy and precision.And virus samples were tested in a preliminary application.Results MAPREC for quantitation of 480-A and 525-C mutants in type I Sabin polio vaccine strain was set up by using CY5 fluorescent labelling.The nominal mutant content of 100% DNA control Standard,IS DNA Standard,LMVR Standard and HMVR Standard are100.00%,2.00%,1.84% and 2.56%,respectively,the tested results were 95.09%,2.06%,1.45% and 1.92%.The tested results of all four International Standard materials were mainly consistent with assigned contents of these references and CV between tests was less than 15%.The mutant rates for working seed of type I Sabin oral poliovirus vaccine,vaccine bulk and a single harvest were between 1.05%—1.22% tested by this method,which met WHO requirements.Conclusion Fluorescent MAPREC was set up and could be used in quantitaion of mutant rate in quality control of type I Sabin polio vaccine strain in replacement of radiolabelling.

关 键 词:聚合酶链式反应和限制性内切酶酶切技术作突变分析 脊髓灰质炎病毒 疫苗 荧光标记 突变率 

分 类 号:R392-33[医药卫生—免疫学]

 

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