Development and preliminary verification of a multiplex RT-qPCR assay for encapsulation efficiency determination of each component of mRNA combination vaccine
10.13200/j.cnki.cjb.004716
- VernacularTitle:mRNA联合疫苗各组分包封率多重RT-qPCR检测方法的建立及初步验证
- Author:
Yiying LIU
- Publication Type:Journal Article
- Keywords:
Encapsulation efficiency;
Multiplex RT-qPCR;
Oligo dT magnetic beads;
mRNA combination vaccines
- From:
Chinese Journal of Biologicals
2026;39(06):679-685+693
- CountryChina
- Language:Chinese
-
Abstract:
Objective To address the challenge of separately detecting the encapsulation efficiency of multiple components in mRNA combination vaccines,a specific detection method based on multiplex RT-qPCR was developed to achieve accurate quality control of the encapsulation efficiency of individual mRNA components in lipid nanoparticles(LNPs).Methods Oligo dT magnetic beads were used to selectively isolate free mRNA from LNPs without disrupting intact LNPs.Specific primers and probes were designed for different mRNA sequences,and a multiplex RT-qPCR assay was established to quantify the content of each component in free mRNA.The linear range,sensitivity,and repeatability of the method were validated.Results The developed multiplex RT-qPCR method could specifically distinguish different mRNA components,overcoming the limitation of traditional RiboGreen dye-based methods that cannot differentiate individual mRNA species.The assay exhibited a broad linear range(102-107 copies) for each mRNA component,with both intra- and inter-assay coefficients of variation(CVs)<2.0%,meeting the requirements for precise quantification.Additionally,the recovery rate of free mRNA enriched by Oligo dT magnetic beads exceeded 75%,and the process did not damage the mRNA-LNP structure.Conclusion A multiplex RT-qPCR-based method for detecting the encapsulation efficiency of mRNA combination vaccines was successfully established,enabling independent quantification of different mRNA components,which provides a reliable tool for process optimization and quality control of multivalent mRNA vaccines.
