多价新型冠状病毒变异株mRNA疫苗组分比例ddPCR检测方法的建立及验证

Establishment and Validation of ddPCR Method for Component Proportion of Multivalent Mutant COVID-19 mRNA Vaccine

关键词:  多价新型冠状病毒变异株mRNA疫苗  组分比例  ddPCR  质量控制

Key words:  multivalent mutant COVID-19 mRNA vaccine  component proportion  ddPCR  quality control

摘要:

目的 建立多价新型冠状病毒变异株mRNA疫苗组分比例ddPCR检测方法并进行验证。方法 以不同新型冠状病毒变异株mRNA为靶基因，选取各自保守序列设计引物和探针，建立多价新型冠状病毒变异株mRNA疫苗组分比例ddPCR检测方法，并进行了线性范围、重复性、准确度、中间精密度、耐用性和适用性等方法学验证。结果 mRNA浓度在1~150 pg·mL^-1内时线性良好；重复性变异系数(coefficient of variation，CV)＜10%；准确度回收率84%~114%，CV＜10%；中间精密度和耐用性CV均＜5%；并且4种不同二价新型冠状病毒mRNA疫苗组份比例检测结果CV均≤5%。结论 建立的多价新型冠状病毒变异株mRNA疫苗组分比例ddPCR检测方法灵敏度高，稳定性好，特异性强，适用于多价新型冠状病毒mRNA疫苗进行组分比例和含量检测。

Abstract:

OBJECTIVE To establish and verify a ddPCR method to determinate the proportion of mRNA components in multivalent mutant COVID-19 mRNA vaccine. METHODS Different types of coronavirus mRNA were used as target genes, and their conserved sequences were selected to design primers and probes. The ddPCR method for measuring the proportion of multivalent mutant COVID-19 mRNA vaccine components was established, and the linear range, repeatability, accuracy, intermediate precision, durability and applicability of the method were verified. RESULTS When the total mRNA concentration ranged from 1 to 150 pg·mL^-1, the linearity was good. Repeatability coefficient of variation(CV) was within 10%. The recoveries of accuracy verification were 84%-114%, CV＜10%. The intermediate precision and durability coefficient of variation CV were all＜ 5%, and the CV of the proportion of components in the four different bivalent COVID-19 mRNA vaccines was all≤5%. CONCLUSION The ddPCR method established for detecting the component proportion of multivalent mutant COVID-19 mRNA vaccine has high sensitivity, good stability and strong specificity, which is suitable for detecting the component proportion and content of multivalent COVID-19 mRNA vaccine.