摘要
目的:建立一种多引物对检测HIV-1病毒的逆转录实时荧光定量PCR(RT-qPCR)方法,用于干细胞产品的病毒安全性质量控制。方法:利用SnapGene软件,选择HIV-1病毒gag-pol基因相对保守位置设计多组模板引物探针,建立RT-qPCR探针法,验证方法的特异性、灵敏度、精密度和耐用性。结果:本方法共设计6组引物探针,可检测到83.0%的HIV-1病毒,hMSCs无明显扩增曲线,加标回收率为70%~130%;检测灵敏度为2 copies·μL^(-1);重复性验证通过检测3个不同拷贝浓度质粒对照品(2×10^(7)、2×10^(5)、2×10^(3)copies·μL^(-1))进行评价,3个浓度的3次重复检测的RSD为2.2%~8.3%;使用不同型号荧光定量PCR系统对上述3个浓度质粒对照品进行耐用性验证,结果显示RSD为0.34%~2.3%。结论:本研究建立的检测HIV-1病毒的方法特异性和重复性好,灵敏度高,经验证可用于生产检定用细胞基质、干细胞制品HIV-1的检测。
Objective:To establish a reverse transcription real-time quantitative PCR(RT-qPCR)method using multiple primer pairs for HIV-1 detection,aiming at enhancing the quality control of viral safety in stem cell products.Methods:Using SnapGene software,multiple sets of template-specific primers and probes were designed targeting relatively conserved regions of the HIV-1 virus gag-pol gene to establish the RT-qPCR probe-based assay.The specificity,sensitivity,precision,and robustness of the method were validated.Results:The assay utilized six primerprobe sets,which demonstrated 83.0%coverage of HIV-1 variants.No detectable amplification curves were observed in human mesenchymal stem cells(hMSCs),and the spiked recovery rates ranged from 70%to 130%.A detection sensitivity of 2 copies·μL^(-1) was achieved.Repeatability was assessed by testing a plasmid standard at three concentrations(2×10^(7) copies·μL^(-1),2×10^(5) copies·μL^(-1),and 2×10^(3) copies·μL^(-1),respectively)in triplicate,with relative standard deviation(RSD)values ranging from 2.2%to 8.3%.Robustness was evaluated across different RT-qPCR platforms,yielding inter-system RSD values of 0.34%-2.3%for the same set of standards.Conclusion:The study develops an HIV-1 detection method that demonstrates superior specificity,excellent repeatability,and high sensitivity,and is validated for testing HIV-1 in biological products such as production-qualified cell substrates and stem cell products.
作者
袁子维
王瑶
李瑶玲
房吉庆
杨英
饶春明
YUAN Zi-wei;WANG Yao;LI Yao-ling;FANG Ji-qing;YANG Ying;RAO Chun-ming(JOINN Pharmaceutical Quality Research and Testing(Beijing)Co.,Ltd.,Beijing 102605,China)
出处
《药物分析杂志》
北大核心
2025年第10期1660-1669,共10页
Chinese Journal of Pharmaceutical Analysis
基金
北京市科技计划课题:基因修饰免疫细胞和基因治疗药物质量控制关键技术与服务平台建设(Z221100007922015)。
