基于多重荧光定量PCR的CHO宿主细胞DNA残留量及片段化程度检测方法的建立及验证  

Establishment and verification of multiplex fluorescence quantitative PCR method for detection of CHO host cell residual DNA content and fragmentation degree

作　　者：杨颢 时景景 周宇荀[1] 李凯[1] 肖君华[1] YANG Hao;SHI Jingjing;ZHOU Yuxun;LI Kai;XIAO Junhua(College of Biological Science and Medical Engineering,Donghua University,Shanghai 201620,China)

机构地区：[1]东华大学生物与医学工程学院,上海201620

出　　处：《中国生物制品学杂志》2024年第12期1495-1504,共10页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31772550);国家重点研发计划(2018YFA081101)。

摘　　要：目的 建立基于多重荧光定量PCR的CHO宿主细胞DNA残留量及片段化程度的检测方法,并进行验证,以期为相关疫苗安全性检测提供可靠的方法。方法 根据CHO细胞Alu序列家族成员clone250和clone49c设计长短2套引物(Alu-L及Alu)及共用探针,同时引入辣椒叶UBI3基因作为外标基因,设计相应引物及探针,建立Alu序列与外标基因的多重反应体系,通过多重荧光定量PCR法对样本中CHO细胞DNA残留量进行定量检测,并验证方法的线性范围、定量限、专属性、耐用性、精密性、准确性。另通过比较Alu长短序列扩增曲线的△Ct[Ct(Alu-L)-Ct(Alu)],判断CHO细胞DNA片段化程度。结果 多重反应标准曲线在0.001~0.1 ng/μL浓度范围内,与Ct值呈良好的线性关系,R~2≥0.99,外标基因的加入不影响对CHO靶标的检测能力;方法的定量限为0.5 fg/μL;人类、E.coli、大鼠、小鼠DNA对检测无影响,针对CHO细胞残留DNA的引物探针在7种动物细胞系基因组中无特异性扩增;碎片化DNA样本不影响检测结果;精密性验证变异系数(CV)均<15%;在含1%BSA的生理盐水和PBS 2种溶剂中模拟样本回收率均在70%~130%范围内。Alu长短片段扩增曲线△Ct随样本碎片化程度的升高而增加,拟合曲线R~2≥0.99。结论本研究建立的多重荧光定量PCR检测方法能够快速准确地对CHO细胞残留DNA进行定量,还可根据△Ct判断样本片段化程度,可用于相关疫苗安全性的检测。Objective To establish and verify a multiplex fluorescence quantitative PCR method for detection of CHO host cell residual DNA content and fragmentation degree,so as to provide a reliable method for safety detection of related vaccines.Methods Two sets of primers(Alu-L and Alu)and universal probe were designed for clone250 and clone49c,members of Alu sequence family of CHO cells.The UBI3 gene of capsicum was introduced as external standard gene,and corresponding primers and probe were designed.Multiplex reaction system of Alu sequence and external standard gene was established,and the residual DNA of CHO cells in samples was quantified by the multiplex fluorescence quantitative PCR.The linear range,limit of quantitation(LOQ),specificity,robustness,precision and accuracy of the method were verified.In addition,the DNA fragmentation degree of CHO cells was determined by comparing the△Ct[Ct(Alu-L)-Ct(Alu)]of the amplification curves of long and short primers of Alu sequences.Results The multiplex reaction standard curve had a good linear relationship in the range of 0.001-0.1 ng/μL,R^(2)≥0.99.The addition of external standard gene had no effect on the detection ability for CHO target,and the LOQ was 0.5 fg/μL.The DNA of human,E.coli,rats and mice had no effect on the detection,the primers targeting residual DNA of CHO cells had no specific amplification in the genomes of seven animal cell lines,and the fragmented DNA samples showed no effect on the detection results.The CVs of precision verification were all less than 15%,and the recoveries of simulated samples in normal saline and PBS containing 1%BSA were in the range of 70%-130%.The△Ct of Alu and Alu-L amplification curves increased with the degree of sample fragmentation,and the fitting curve R~2≥0.99.Conclusion The multiplex fluorescence quantitative PCR detection method established in this study can rapidly and accurately quantify the residual DNA of CHO cells and determine the fragmentation degree of samples according to△Ct,which can be used for t

关 键 词：宿主细胞残留DNA CHO细胞 TAQMAN探针 多重荧光定量PCR法 

分 类 号：R34[医药卫生—基础医学]