摘要
目的构建实时荧光定量PCR(qPCR)方法,用于细胞治疗产品的快速微生物检测。方法以人脐带间充质干细胞、诱导多能干细胞、NK细胞及T细胞四种代表性细胞治疗产品为对象,系统验证qPCR微生物6 h内快速检测方法,并与《中国药典》中的传统培养法进行平行比较。结果在理想样本条件下,qPCR法对金黄色葡萄球菌、铜绿假单胞菌等7种药典推荐标准菌株的检测限均≤100 CFU/mL,理论可达≤25 CFU/mL;四种真实细胞样本经加标实验后,qPCR方法与培养法一致性良好,但高浓度细胞因子或细胞本身可致细菌检测循环阈值(Ct值)后移1~3个循环,需通过离心并重悬或稀释预处理消除抑制;各基质内标Ct值(VIC Ct)稳定在30±1.5,变异系数(CV)<5%,方法重现性良好。结论经验证优化后的qPCR方法可在6 h内完成细胞治疗产品的无菌检查,满足法规检测限(LOD)≤100 CFU/mL的要求,为细胞制品的快速放行及库存风险控制提供了可行、合规的新路径。
Objective Establish a real-time fluorescence quantitative PCR(qPCR)method for rapid microbial detection in cell therapy products.Methods Four representative cell therapy products,including human umbilical cord mesenchymal stem cells,induced pluripotent stem cells,NK cells,and T cells,were selected as the objects.The rapid microbial detection method based on real-time fluorescence quantitative PCR within 6 hours was systematically verified and compared with the traditional culture method in the Chinese Pharmacopoeia.Results Under ideal sample conditions,the detection limit of qPCR for seven standard strains recommended by the pharmacopoeia,such as Staphylococcus aureus and Pseudomonas aeruginosa,was≤100 CFU/mL,and theoretically could reach≤25 CFU/mL.After spiking experiments on four real cell samples,the qPCR method showed good consistency with the culture method,but high concentrations of cytokines or cells themselves could cause a 1-3 cycle shift in the cycle threshold(Ct value)of bacterial detection,which could be eliminated by centrifugation and resuspension or dilution pretreatment.The Ct values of the internal standards(VIC Ct)in each matrix were stable at 30±1.5,with the coefficient of variation(CV)<5%,indicating good method reproducibility.Conclusion The optimized qPCR method can complete the sterility test of cell therapy products within 6 hours,meeting the regulatory LOD≤100 CFU/mL requirement,and provides a feasible and compliant new path for the rapid release and inventory risk control of cell products.
作者
宋丹
史静静
卜修民
赵正阳
SONG Dan;SHI Jingjing;BU Xiumin(Center for Inspection of Jiangsu Medical Products Administration,Nanjing 210019,China;不详)
出处
《中国处方药》
2025年第23期33-37,共5页
Journal of China Prescription Drug
基金
江苏省药品监督管理局科学科研计划项目(202230)。
关键词
实时荧光定量PCR
快速微生物检测
细胞治疗产品
Real-time fluorescence quantitative PCR
Rapid microbial detection
Cell therapy products
