嵌合抗原受体T(CAR-T)预示着癌症免疫治疗领域的范式转变。逆转录病毒介导的基因转移用于在癌症治疗中的一系列基础研究和临床竞赛中将特异性CAR表达盒递送到T细胞中。然而,有必要设计一种针对不同CAR构建体的精确且经过验证的定量方法.在调查中,建立了TaqMan实时qPCR方法,利用引物打靶的φ基因序列。这种方法提供了一种快速的,敏感,可重复,以及在整合DNA水平上评估逆转录病毒拷贝数的准确工具。重要的是,所建立的qPCR不显示与非转导的T细胞或组织的交叉反应性。表征TaqMan实时PCR动力学的回归方程为y=-3.3841x41.402(R2=0.999),扩增效率为97.47%。值得注意的是,所建立的qPCR方法实现了43.1拷贝/μL的最小检测。此外,组内和组间差异均低于4%,强调了所建立方法的良好重复性。我们的体外和体内结果也支持其敏感性,特异性,和稳定性。因此,这种方法为研究人员提供了一种经济有效的工具,可以在体外和体内量化CAR拷贝.
Chimeric-antigen-receptor-T (CAR-T) have heralded a paradigm shift in the landscape of cancer immunotherapy. Retrovirus-mediated gene transfer serves to deliver the specific CAR expressing cassette into T cells across a spectrum of basic research and clinical contests in cancer therapy. However, it is necessary to devise a precise and validated quantitative methodology tailored to the diverse CAR constructs. In the investigation, a TaqMan real-time qPCR method was developed, utilizing primers targeting ψ gene sequence. This method offers a swift, sensitive, reproducible, and accurate tool for evaluating retroviral copy numbers at the integrated DNA level. Importantly, the established qPCR exhibits no cross-reactivity with non-transduced T cells or tissues. The regression equation characterizing TaqMan real-time PCR dynamics is y = -3.3841x + 41.402 (R2 = 0.999), showing an amplification efficiency of 97.47 %. Notably, the established qPCR method achieves a minimum detection of 43.1 copies/μL. Furthermore, both intra- and inter-group discrepancies remain below 4 %, underscoring the good repeatability of the established method. Our in vitro and in vivo results also support its sensitivity, specificity, and stability. Consequently, this method offers researchers with a cost-effective tool to quantify CAR copies both in vitro and in vivo.