诺如病毒基因工程疫苗中残余宿主DNA实时荧光定量PCR检测方法的建立及验证  

Development and validation of real-time fluorescent quantitative PCR method for determination of residual host DNA in recombinant norovirus vaccine

作　　者：姜英[1] 雷清[1] 张巧玲 陈继军[1] 杨俊杰[1] JIANG Ying;LEI Qing;ZHANG Qiao-ling;CHEN Ji-jun;YANG Jun-jie(The Fourth Department of Research,Lanzhou Institute of Biological Products,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所第四研究室,甘肃兰州730046

出　　处：《中国生物制品学杂志》2020年第12期1426-1430,共5页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2018ZX09739002-005)。

摘　　要：目的建立汉逊酵母表达的重组诺如病毒基因工程疫苗中残余宿主DNA实时荧光定量PCR(real-time quantitative PCR,qPCR)检测方法,并进行验证。方法采用磁珠法提取汉逊酵母基因组DNA,获得标准品,建立q PCR标准曲线,并进行专属性、线性、准确度、精密度和耐用性验证。结果专属性验证结果显示,与对照制剂(注射用水、缓冲液、Vero细胞和CHO细胞培养上清DNA)相比,建立的方法对汉逊酵母DNA具有特异性扩增曲线;线性验证中5次试验标准曲线相关系数(R2)均>0.98;准确度验证试验不同浓度样品的加标回收率在95.12%~105.72%之间,均在70%~130%范围内;重复性和中间精密度验证试验相对标准偏差(RSD)均小于30%;耐用性验证中,Proteinase K不同消化温度下检测结果的RSD为20.75%,不同蛋白浓度供试品检测结果的RSD为27.11%,均符合《中国药典》三部(2015版)要求。结论建立的方法专属性、线性、准确度、精密度和耐用性均符合要求,可用于检测重组诺如病毒类病毒颗粒(virus-like particle,VLP)疫苗中残余宿主DNA。Objective To develop and validate a real-time fluorescent quantitative PCR(qPCR)method for determination of residual host DNA in recombinant norovirus vaccine expressed in Hansenula polymorpha.Methods Genomic DNA of H.polymorpha was extracted with magnetic beads and used as a DNA reference,and the standard curve for qPCR was established and validated for specificity,linearity,accuracy,precision and durability.Results Specify amplification curve of qPCR was obtained by using H.polymorpha DNA rather than the controls(water for injection,buffer as well as DNAs from culture supernatant of Vero cells and CHO cells).All the correlation coefficient(R2)in 5 validation tests for linearity were more than 0.98.The recovery rates of spiked sample at various concentrations were 70%~130%in accuracy validation test.The RSDs in validation tests for reproducibility and intermediate precision were less than 30%.However,in the durability test,the RSD of determination results of samples digested with Proteinase K at various temperatures was 20.75%,while that of samples at various protein concentrations was 27.11%,which met the requirements in Chinese Pharmacopoeia(VolumeⅢ,2015 edition).Conclusion The specificity,linearity,precision,accuracy and durability of the developed could met the relevant requirements,indicating that the method may be used for the determination of residual DNA in norovirus VLP vaccine.

关 键 词：残余宿主DNA 实时荧光定量PCR 重组诺如病毒VLP疫苗 汉逊酵母 

分 类 号：R373.1[医药卫生—病原生物学]