重组新型冠状病毒疫苗(腺病毒载体)病毒颗粒数微滴式数字PCR方法的建立  

Establishment of droplet digital PCR for quantification of virus particles in recombinant adenovirus-vectored SARS-CoV-2 vaccine

作　　者：赵丹华 彭沁桦 李玉华[1] 吴小红[1] Zhao Danhua;Peng Qinhua;Li Yuhua;Wu Xiaohong(National Institutes for Food and Drug Control,Division of Arboviral Vaccines,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院虫媒病毒疫苗室,北京102629

出　　处：《中华微生物学和免疫学杂志》2023年第11期881-885,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家重点研发计划(2021YFC2302404)。

摘　　要：目的构建一种重组新型冠状病毒疫苗(腺病毒载体)病毒颗粒数微滴式数字PCR(droplet digital PCR,ddPCR)检测方法。方法以新型冠状病毒BA.1株基因组为靶基因,选取刺突蛋白保守序列设计引物和探针,建立重组新型冠状病毒疫苗病毒颗粒数ddPCR方法,并进行线性、准确度、专属性、重复性和耐用性等方法学验证。结果ddPCR的最佳退火温度是63℃,病毒颗粒数为5×10^(5)~2×10^(7) VP/ml时,线性和回收率良好,探针和引物特异性好,重复性和中间精密度变异系数(coefficient of variation,CV)都在10%以内,耐用性CV值在15%以内,并且与qPCR结果相比,CV值都在10%以内。结论本研究建立的ddPCR方法灵敏度高、稳定性好、特异性强,可用于重组新型冠状病毒疫苗(腺病毒载体)病毒颗粒数的定量检测。ObjectiveTo construct a droplet digital PCR(ddPCR)for quantification of virus particles in recombinant adenovirus-vectored SARS-CoV-2 vaccine.MethodsThe genome of SARS-CoV-2 BA.1 strain was used as the target gene.A set of primer and probe was designed based on the conserved sequence of spike protein,and the ddPCR for quantification of virus particles in recombinant adenovirus-vectored SARS-CoV-2 vaccine was established.The linearity,accuracy,specificity,repeatability and durability of the method were verified.ResultsThe optimal annealing temperature for ddPCR was 63℃.When the number of virus particles was in the range of 5×10^(5)-2×10^(7) VP/ml,the linearity and the recovery rate of the method were good;the specificity of the probe and primer was good;the coefficient of variation(CV)values of the repeatability and the intermediate precision were within 10%;the CV value of the durability was within 15%.When comparing ddPCR with qPCR,the CV values were all within 10%.ConclusionsThe established ddPCR had high sensitivity,good stability and strong specificity,suggesting that it could be used for the quantitative detection of virus particles in recombinant adenovirus-vectored SARS-CoV-2 vaccine.

关 键 词：重组新型冠状病毒疫苗(腺病毒载体) 病毒颗粒数 ddPCR 质量控制 

分 类 号：R511[医药卫生—内科学] R440[医药卫生—临床医学]