rAAV5基因组滴度测定的数字PCR方法研究  

Study on digital PCR methods for rAAV5 genomic titer determination

作　　者：郑红梅 秦玺[1] 李永红[1] 于雷[1] 毕华[1] 饶春明[1] 朱留强 杨靖清[1] 史新昌[1] 周勇[1] ZHENG Hong-mei;QIN Xi;LI Yong-hong;YU Lei;BI Hua;RAO Chun-ming;ZHU Liu-qiang;YANG Jing-qing;SHI Xin-chang;ZHOU Yong(Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京100050

出　　处：《药物分析杂志》2023年第11期1826-1832,共7页Chinese Journal of Pharmaceutical Analysis

基　　金：北京市科学技术委员会资助(Z221100007922015)。

摘　　要：目的:建立并验证重组5型腺相关病毒(recombinant adeno-associated virus type 5,rAAV5)基因组滴度测定的数字PCR方法,并对微滴数字PCR方法和芯片数字PCR方法进行比较。方法:分别用不同浓度的SDS和EDTA前处理r AAV5样品,用微滴数字PCR测其基因组滴度,对病毒样品前处理试剂进行优化;在其他试验条件不变的情况下,分别设置微滴数字PCR的退火温度为60、58、54、52℃,以WPRE引物扩增,比较扩增效果,对数字PCR的退火温度进行优化;分别采用微滴数字PCR和芯片数字PCR测定rAAV5的基因组滴度并比较。结果:终浓度为1%SDS和62.5 mmol·L^(-1)EDTA按1∶1(v/v)的比例混合后前处理rAAV5,测定的基因组拷贝数较高,前处理液裂解效果较好;当退火温度为54℃时,微滴数字PCR中阳性微滴和阴性微滴分离效果最好,扩增特异性最强;2种测定结果的试验室内精密度和回收率接近,测定同一裂解样本的结果较为接近(在1个数量级)。结论:初步建立了数字PCR方法并验证,为后续的进一步验证奠定基础。Objective:To establish and verify a digital PCR method for the determination of recombinant adeno-associated virus type 5(rAAV5)genome titer,and to compare the performance of chip and droplet digital PCR methods.Methods:By examine the different lysing outcome from various concentrations of SDS or EDTA,the adeno-associated virus lysis method was optimized.To ensure other experimental conditions being consistent,the rAAV5 sample was amplified with WPRE primers in the droplet digital PCR method,and the annealing temperature was set to 60,58,54,52℃.With amplification effect compared,the annealing temperature was optimized.Both chip and droplet digital PCR were tested to determine the genomic titer of rAAV5,and the consistency of the results of the two methods was compared.Results:The final concentration of 1%SDS and 62.5 mmol·L^(-1) EDTA was mixed 1:1(v/u)and then rAAV5 was lysed.The measured genome copy number was high,the lysing outcome was good.At annealing temperature 54℃,the positive droplets in the droplet digital PCR and the separation effect of negative droplets was ideal,and the amplification specificity is the strongest.Comparing the two methods,it was found that the laboratory precision and recovery rates of the two determination results are similar,and the results of determining the same pyrolysis sample were relatively close(on the same order of magnitude).Conclusion:A preliminary digital PCR method has been established and validated,laying the foundation for further validation in the future.

关 键 词：重组5型腺相关病毒 基因组滴度 微滴数字PCR 芯片数字PCR 基因治疗 

分 类 号：R917[医药卫生—药物分析学]