Ren Jinfeng, Liu Ke, Hu Lang, Yang Ruoning, Liu Yuting, Wang Siyu, Chen Xinzhu, Zhao Shuli, Jing Luyao, Liu Tiantian, Hu Bin, Zhang Xuefeng, Wang Hui, Li Hui
Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogenic Biology and Immunology, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China.
National Experimental Demonstration Center for Basic Medicine Education, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China.
Mol Biotechnol. 2025 Mar;67(3):1156-1164. doi: 10.1007/s12033-024-01115-8. Epub 2024 Mar 8.
Precise quantification of human cells in preclinical animal models by a sensitive and specific approach is warranted. The probe-based quantitative PCR (qPCR) assay as a sensitive and swift approach is suitable for the quantification of human cells by targeting human-specific DNA sequences. In this study, we developed an efficient qPCR assay targeting human-specific DNA in ST6GALNAC3 (termed ST6GAL-qPCR) for the quantification of human cells in preclinical animal models. ST6GAL-qPCR probe was synthesized with FAM and non-fluorescent quencher-minor groove binder conjugated to the 5' and 3' end of the probe, respectively. Genomic DNA from human, rhesus monkeys, cynomolgus monkeys, New Zealand White rabbits, SD rats, C57BL/6, and BALB/c mice were utilized for analyzing the specificity and sensitivity of the ST6GAL-qPCR assay. The ST6GAL-qPCR assay targeted human-specific DNA was cloned to pUCM-T vector and released by EcoR I/Hind III digestion for generating a calibration curve. Cell mixing experiment was performed to validate the ST6GAL-qPCR assay by analysis of 0.1%, 0.01%, and 0.001% of human leukocytes mixed with murine thymocytes. The ST6GAL-qPCR assay detected human DNA rather than DNA from the tested animal species. The amplification efficiency of the ST6GAL-qPCR assay was 93% and the linearity of calibration curve was R = 0.999. The ST6GAL-qPCR assay detected as low as 5 copies of human-specific DNA and is efficient to specially amplify as low as 30-pg human DNA in the presence of 1 μg of DNA from the tested species, respectively. The ST6GAL-qPCR assay was able to quantify as low as 0.01% of human leukocytes within murine thymocytes. This ST6GAL-qPCR assay can be used as an efficient approach for the quantification of human cells in preclinical animal models.
需要采用灵敏且特异的方法对临床前动物模型中的人类细胞进行精确量化。基于探针的定量聚合酶链反应（qPCR）检测作为一种灵敏且快速的方法，通过靶向人类特异性DNA序列适用于人类细胞的量化。在本研究中，我们开发了一种高效的qPCR检测方法，靶向ST6GALNAC3中的人类特异性DNA（称为ST6GAL-qPCR），用于临床前动物模型中人类细胞的量化。ST6GAL-qPCR探针的合成是将FAM与非荧光淬灭剂-小沟结合物分别连接到探针的5'和3'末端。利用来自人类、恒河猴、食蟹猴、新西兰白兔、SD大鼠、C57BL/6和BALB/c小鼠的基因组DNA分析ST6GAL-qPCR检测的特异性和灵敏度。将靶向人类特异性DNA的ST6GAL-qPCR检测克隆到pUCM-T载体中，并通过EcoR I/Hind III消化释放以生成校准曲线。进行细胞混合实验，通过分析与小鼠胸腺细胞混合的0.1%、0.01%和0.001%的人类白细胞来验证ST6GAL-qPCR检测。ST6GAL-qPCR检测可检测到人类DNA，而非来自受试动物物种的DNA。ST6GAL-qPCR检测的扩增效率为93%，校准曲线的线性为R = 0.999。ST6GAL-qPCR检测分别能够检测低至5拷贝的人类特异性DNA，并且在存在1μg受试物种DNA的情况下能够高效地特异性扩增低至30 pg的人类DNA。ST6GAL-qPCR检测能够在小鼠胸腺细胞中量化低至0.01%的人类白细胞。这种ST6GAL-qPCR检测可作为临床前动物模型中人类细胞量化的一种有效方法。
