基于逆转录酶活性的复制型慢病毒检测方法的建立及应用  

Establishment and application of a reverse transcriptase activity assay for replication-competent lentivirus detection

作　　者：房恩岳 常宇桐 何畦嘉 潘一西 胡孔新[1] FANG Enyue;CHANG Yutong;HE Qijia;PAN Yixi;HU Kongxin(Institute of Health Inspection and Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100176,China)

机构地区：[1]中国检验检疫科学研究院卫生检验与检疫研究所,北京100176

出　　处：《中国医药生物技术》2025年第1期64-72,共9页Chinese Medicinal Biotechnology

基　　金：中国检验检疫科学研究院基本科研业务费项目(2023JK004)。

摘　　要：目的建立基于逆转录酶活性的复制型慢病毒检测方法,并进行方法学验证及适用性研究。方法以莫洛尼鼠白血病病毒逆转录酶为标准品,以噬菌体MS2 RNA为模板,经反转录后采用TaqMan探针实时荧光定量PCR方法检测特异性扩增信号。对方法的专属性、线性范围、准确度、重复性、中间精密度、最低定量限、最低检出限、耐用性和适用性进行验证。结果通过优化qPCR引物、探针浓度及反应条件,已建立稳定灵敏的逆转录酶活性检测方法。该方法专属性良好,对4种类型的细胞上清样品均无特异性扩增;在10^(4)~10^(9)pU/μL之间具有良好线性范围,r^(2)>0.99,且扩增效率处于93%~97%;准确度回收率在86%~102%,重复性RSD≤6%;中间精密度良好,加标样品RSD≤4%;最低定量限为8000 pU/μL;最低检出限为100 pU/test;耐用性良好,RSD≤8%,回收率89%~105%;通过指示细胞培养法接种的三类样品(CAR-T细胞终产品样品、慢病毒载体生产终末细胞样品、慢病毒载体未加工收获液)及其病毒感染加标样品,在感染终末阶段收样后使用本方法进行逆转录酶活性检测的结果显示,三类样品均未检出复制型慢病毒,而病毒感染加标组样品均可检出病毒,方法适用性良好。结论建立了稳定可靠的基于逆转录酶活性的复制型慢病毒检测方法,各项方法学验证结果均达满意要求,且方法适用于指示细胞培养法终末阶段收样的检测,为基因治疗产品临床应用的安全性研究提供技术支撑。Objective To establish a reverse transcriptase activity assay for the detection of replication-competent lentivirus(RCL)and to validate the methodology.Methods The RNA of bacteriophage MS2 served as a template for reverse transcription,with specific amplification signals detected via TaqMan-based quantitative Real-time PCR(qPCR).Moloney murine leukemia virus reverse transcriptase(M-MLVRT)was used as a standard for quantitative assay by plotting a standard curve.Methodological parameters,including specificity,dynamic range,precision,repeatability,intermediate precision,limit of quantification,limit of detection,robustness and applicability were validated.Results Through the optimization of primers,probe concentration,and reaction conditions,a stable and sensitive assay for reverse transcriptase activity was established,demonstrating high specificity and non-specific amplification across four types of cell supernatant samples.The assay exhibited a wide linear range from 10^(4)to 10^(9)pU/μL,with r^(2)value higher than 0.99,and the amplification efficiency ranged from 93%to 97%.The accuracy(rate of recovery)fell between 86%and 102%,with repeatability(relative standard deviation,RSD)less than or equal to 6%.Intermediate precision and robustness RSD were less than or equal to 4%and 8%,respectively,and robustness recovery ranged from 89%to 105%.The limit of quantification(LOQ)and limit of detection(LOD)were determined as 8000 pU/μL and 100 pU/test,respectively.Reverse transcriptase activity was successfully detected in the terminal stage samples of three cell culture assay types-CAR-T cells,end of production cell(EOPC),and unprocessed bulk(UPB)-as well as their virus-infected samples.Notably,no activity was detected in the three sample types without virus infection,while the virus-infected spiked samples were successfully detected.Conclusions The reverse transcriptase activity-based RCL assay was successfully established,with all validation results meeting detection requirements.It is suitable for detecting terminal

关 键 词：复制型慢病毒 逆转录酶 嵌合抗原受体T细胞 方法学验证 

分 类 号：Q812[生物学—生物工程]