重组腺相关病毒装载目的核酸完整性分析  

Analysis on integrity of target nucleic acid in recombinant adeno-associated virus

作　　者：黄天治 秦玺[2] 于雷[2] 郑祖亮 郭林 史新昌[2] 张怡轩[1] HUANG Tianzhi;QIN Xi;YU Lei;ZHENG Zuliang;GUO Lin;SHI Xinchang;ZHANG Yixuan(不详;School of Life Sciences and Biopharmaceuticals,Shenyang Pharmaceutical University,Shenyang 117004,Liaoning Province,China)

机构地区：[1]沈阳药科大学生命科学与生物制药学院,辽宁沈阳117004 [2]中国食品药品检定研究院国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室国家药品监督管理局生物制品质量研究与评价重点实验室,北京100050 [3]北京深蓝云生物科技有限公司,北京102600

出　　处：《中国生物制品学杂志》2024年第6期641-645,共5页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2023YFC3403305);中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2023-PT350-01)。

摘　　要：目的探讨重组腺相关病毒(recombinant adeno-associated virus,rAAV)装载目的核酸完整性检测方法,并初步建立分析算法。方法消化rAAV外游离DNA片段,提取病毒基因组,设计5对搭接引物,采用正交排列方式,分别用数字PCR仪进行检测,常规条件采用EveGreen和模板4μL的20μL反应体系,数字PCR条件:95℃5 min;95℃15 s,55℃30 s,72℃90 s,45个循环。超长核酸片段增强条件采用Mix B-2000 bp和模板2μL的25μL反应体系,采用仪器附带软件定量。最小二乘法拟合分析共有的全长片段数量,进而解读目的核酸完整性分布情况。结果引物间距离不大于1200 nt的片段可得到有效扩增,经系统分析得到一系列片段长约1000 nt的有效片段拷贝数,最小二乘法拟合分析共有片段拷贝数量,估计样本中全长片段不多于1234 copies/μL,该值与测得的最大值(1443 copies/μL)之间差异有统计学意义(P<0.05),差异占比约16.9%。结论初步建立了rAAV装载目的核酸完整性的检测方法,并分析出供试品中存在一定数量的不完整目的核酸,为后续深入研究奠定了基础。Objective To explore a method for detecting the integrity of target nucleic acid in recombinant adeno-associated virus(rAAV),and establish a preliminary analysis algorithm.Methods The free DNA fragment of rAAV was digested,and the virus genome was extracted.Five pairs of overlapping primers were designed,using the orthogonal array method,which were detected by digital PCR respectively,with conventional conditions:20μL reaction system with EveGreen and4μL template,and digital PCR conditions:95℃5 min;95℃15 s,55℃30 s,72℃90 s for 45 cycles.The enhancement condition of ultra-long nucleic acid fragment was 25μL reaction system with Mix B-2000 bp and 2μL template,and the quantitative analysis was performed by using the software attached to the instrument.Using the least square method,the number of full-length fragments was fitted and analyzed,and then the integrity distribution of target nucleic acid was interpreted.Results The fragments with the distance between primers of no more than 1200 nt were amplified effectively,and a series of effective copies of fragments with a length of about 1000 nt were obtained by systematic analysis.The copy number of common fragments was fitted and analyzed by the least square method.It was estimated that the full-length fragments in the sample were no more than 1234 copies/μL,and there was a signficant difference(P<0.05)between this value and the maximum measured value of 1443 copies/μL,with the difference of approximately 16.9%.Conclusion A preliminary detection method for the integrity of target nucleic acid in rAAV has been developed,and a certain amount of incomplete target nucleic acids were analyzed in the test sample,laying a foundation for further in-depth research.

关 键 词：重组腺相关病毒 核酸 完整性 数字PCR 重叠 

分 类 号：R917[医药卫生—药物分析学]