To establish a fluorescence quantitative PCR (qPCR) assay for detecting Torque teno virus (TTV) in human blood and evaluate its role in assessing immune function in kidney transplant recipients.

Based on the TTV standard nucleic acid sequence published in the NCBI (the national center for biotechnology information) database, primers and probes were designed for the UTR region.This region was artificially synthesized and integrated into a plasmid as a detection standard. Reaction conditions, including annealing temperature, primer concentration, and probe concentration, were optimized to establish a standard curve for TTV qPCR detection. Clinical blood samples from 108 kidney transplant recipients and candidates awaiting transplantation were collected from the Transplant Medicine Center of Wuhan University and divided into stable, rejection, pre-transplant, and infection groups based on clinical status. TTV load was measured, and the diagnostic value of TTV copy number for post-transplant infection and rejection was evaluated using receiver operating characteristic (ROC) curve analysis.

The optimized qPCR conditions were determined as an annealing temperature of 56 ℃, with 1 μL each of primers and probes. Under these conditions, a standard curve was generated using standard templates ranging from 9 to 3 log copies/μL, yielding a coefficient of determination R²=0.999, slope=-3.320, intercept=42.28, amplification efficiency=100.1%, and a lower detection limit of 1 log copies/μL. qPCR testing of 108 patient plasma samples revealed correlations between TTV load and infection or rejection. The TTV load in the stable group was 5.125 (4.550, 5.491) log copies/μL, increased in the infection group to 5.888 (5.135, 6.506) log copies/μL, decreased in the rejection group to 4.167 (3.459-4.869) log copies/μL, and was lowest in the pre-transplant group at 2.885 (2.636, 3.233) log copies/μL, with statistically significant differences (P<0.05). The area under the ROC curve (AUC) for TTV load in diagnosing rejection was 0.7875 (P<0.05), with an optimal cutoff value of 4.999 log copies/μL at the maximum Youden index. For diagnosing infection, the AUC was 0.7458 (P<0.05), with an optimal cutoff value of 5.770 log copies/μL.

The established TTV qPCR assay demonstrates good sensitivity and specificity for detecting TTV in human whole blood. A TTV load below 9.98×10⁴ copies/μL indicates a higher risk of rejection, while a load above 5.89×10⁵ copies/μL suggests a higher risk of infection.