荧光定量PCR方法在脊髓灰质炎病毒突变分析中的应用  

Application of fluorescent quantitative PCR method to analysis of poliovirus revertant

作　　者：王潇潇[1] 刘宇[1] 温智恒[1] 杨永娟[1] 李懿[1] 张中洋[1] 李秀玲[1] 

机构地区：[1]北京生物制品研究所有限责任公司,北京101111

出　　处：《中国生物制品学杂志》2014年第12期1579-1587,1594,共10页Chinese Journal of Biologicals

基　　金：科技部863计划(2012AA02A401);国药新产品基金(2013SW22);重大新药创制(2013ZX09402302);北京市科委基金资助基金项目:(Z131100006513007)

摘　　要：目的建立检测脊髓灰质炎病毒突变比例的荧光定量PCR方法,并进行验证及初步应用。方法分别建立检测Ⅰ、Ⅱ、Ⅲ型脊髓灰质炎病毒突变株和非突变株的荧光定量PCR方法,并进行特异性、重复性和灵敏度验证。通过Ct值计算样品的突变比例（revertant proportion,RP）,并进行准确性和重复性验证。采用建立的荧光定量PCR方法检测脊髓灰质炎病毒减毒株的突变比例。结果建立的检测Ⅰ、Ⅱ、Ⅲ型脊髓灰质炎病毒突变株和非突变株的荧光定量PCR方法能够特异性扩增相应基因片段;重复性试验Ct值的变异系数（CV）为0.085%-5.564%;灵敏度为1×10^-7-1×10^-6ng,Ⅰ、Ⅱ、Ⅲ型非突变荧光定量PCR可分别检测到含量约0.084 3、0.029 2和0.266 7 CCID50的病毒。荧光定量PCR测定RP的方法经验证发现,Ⅰ、Ⅱ、Ⅲ型混合质粒对照的RP检测值与理论值的差异较小,分别为0.040 9±0.037 4、0.008 8±0.034 3和-0.029 6±0.026 8。Ⅰ、Ⅱ、Ⅲ型脊髓灰质炎减毒株的RP分别为0.001 0、0.002 0和0.001 7。结论荧光定量PCR分析脊髓灰质炎病毒突变比例的方法,特异性、重复性及准确性均较好,并具有较高的灵敏度,可用于实验室内部对脊髓灰质炎病毒的突变比例测定,并可能成为脊髓灰质炎病毒体内或体外神经毒力评价的替代方法。Objective To develop,verify and preliminarily apply a fluorescent quantitative PCR（QPCR） method for determination of revertant proportion of mutant in poliovirus. Methods QPCR assays for detection of nonrevertant and revertant of poliovirus types Ⅰ,Ⅱ and Ⅲ were developed and verified for specificity,reproducibility and sensitivity. The revertant proportion（RP） of samples was calculated by using Ct value,and the method was verified for accuracy and reproducibility. Results The target genes of nonrevertant and revertant strains of poliovirus types Ⅰ,Ⅱ and Ⅲ were amplified by the developed QPCR. The coefficient of variation（CV） of Ct value in reproducibility test was 0. 085% - 5. 564%.The sensitivity of developed method was 1 × 10^-7- 1 × 10^-6ng. The lowest detection limits of QPCR assays for nonrevertant strains of poliovirus types Ⅰ,Ⅱ and Ⅲ were 0. 084 3,0. 029 2 and 0. 266 7 CCID50 respectively. The differences of theoretical and measured RPs of constructed recombinant nonrevertant and revertant plasmid mixtures of types Ⅰ,Ⅱand Ⅲ were 0. 040 9 ± 0. 037 4,0. 008 8 ± 0. 034 3 and-0. 029 6 ± 0. 026 8 respectively. The RPs of attenuated seed stocks of poliovirus types Ⅰ,Ⅱ and Ⅲ were 0. 001 0,0. 002 0 and 0. 001 7 respectively. Conclusion The developed QPCR method for analysis of mutant of poliovirus showed high specificity,reproducibility,accuracy and sensitivity,which might be used for in-house determination of revertant proportion,and was a potential alternative approach of in vivo or in vitro neurovirulence tests for poliovirus.

关 键 词：脊髓灰质炎病毒 疫苗 荧光定量PCR 突变比例 

分 类 号：R373.22[医药卫生—病原生物学] Q789[医药卫生—基础医学]