An Efficient Probe-Based Quantitative PCR Assay Targeting Human-Specific DNA in ST6GALNAC3 for the Quantification of Human Cells in Preclinical Animal Models.

Abstract

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 R² = 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.