Establishment of reference measurement procedure and reference material for Treponema pallidum

Stem cell preparations, as a new type of biotherapeutic product, should be subject to strict quality control in terms of cell safety. The testing of stem cell donors and blood products used in stem cell cultures, including but not limited to Treponema pallidum, is needed to reduce the risk of transmission of infectious diseases by stem cell medical products. In this study, a reference measurement procedure (RMP) was established based on digital PCR (dPCR). A homogeneous reference material (RM) of TP containing the tpp47 gene has been developed and characterized. Two dPCR assays (A and B) show ideal linearity within five orders of magnitude. The limit of quantification (LoQ) for both assays is 57 copies/reaction; the limits of detection (LoD) are 9.69 and 9.59 copies/reaction, respectively. The quantitative results of the established duplex dPCR assay are in good agreement. The RM of TP containing the tpp47 gene has been developed and characterized. The reference value with its expanded uncertainty is (2.21 ± 0.22) × 106 copies per μL determined by the established dPCR RMP. The developed dPCR was validated by applying a simulated stem cell matrix, and no impact was observed on the accuracy of dPCR. By providing an accurate reference value for the absolute copy number of the target gene, the developed RM can be used to improve the reliability of TP testing in the production of stem cell preparations and clinical diagnostics.