定量检测Ⅲ型Sabin株脊髓灰质病毒突变的荧光MAPREC法的建立和应用  

Establishment and application of fluorescent MAPREC assay for quantitative analysis of mutants in type Ⅲ Sabin poliovirus

作　　者：徐康维[1] 张平 英志芳[1] 何蕊 王剑锋[1] 江征[1] 李长贵[1] 

机构地区：[1]中国食品药品检定研究院,北京100050 [2]北京天坛生物制品有限公司,北京100176

出　　处：《药物分析杂志》2018年第2期288-292,共5页Chinese Journal of Pharmaceutical Analysis

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

摘　　要：目的:使用荧光标记代替同位素标记,建立定量检测Ⅲ型Sabin株脊髓灰质炎病毒472位U到C突变比例的MAPREC(mutant analysis by PCR and restriction enzyme cleavage)法。方法:提取病毒样品RNA,反转录合成c DNA后进行非对称PCR反应以生成单链DNA扩增产物,之后采用荧光标记引物进行扩增,合成双链产物,以突变位点敏感的限制性内切酶作用后电泳分离,并分析样品的突变比例。结果:采用建立的方法对4个国际标准品进行检测,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%、0.9%、0.7%、1.1%,检测结果分别为95.59%、0.84%、0.68%、1.07%,与其标示值一致性良好,并且不同实验间RSD均小于15%。检测9个批次样品,结果突变率均小于1%,符合WHO要求。结论:初步建立了基于荧光素标记的MAPREC方法,可以代替同位素法进行病毒突变的定量检测。Objective：To avoid the usage of the radio-active isotope in traditional MAPREC（mutant analysis by PCR and restriction enzyme cleavage） method,and a modified method based on fluorescence labelling will be established to test the content of mutant at 472 from U to C in Sabin poliovirus. Methods：Viral RNA was extracted and used for c DNA synthesis,then the unsymmetrical PCR was applied to generate single-stranded DNA. A primer labelled with fluorescein was used in a double-stranded DNA synthesis through one-step DNA polymerase reaction. The labelled double-stranded products were then digested with restriction enzyme and separated by electrophoresis. The mutant content was calculated. Results：Four international standards materials including 100% DNA control standard,IS DNA standard,LMVR standard and HMVR standard were tested by this method. The test results were 95.59%,0.84%,0.68% and 1.07%,showing satisfying consistency with the labelled contents,which are 100%,0.9%,0.7% and 1.1 %. RSDs among different experiments were less than 15%. The mutant contents of 9 lots of samples tested by this method were all less than 1%,which met WHO requirements. Conclusion：Fluorescent MAPREC method was successfully established,which could be an alternative for isotopic method in quantitative analysis of mutants in virus.

关 键 词：MAPREC 脊髓灰质炎病毒 疫苗 荧光标记 突变率 

分 类 号：R917[医药卫生—药物分析学]