构建人全血细环病毒(TTV)的荧光定量PCR(qPCR)检测方法，并评价其在肾移植受者免疫功能评估中的作用。

根据美国国家生物信息中心数据库中公布的TTV标准核酸序列，针对非翻译区设计引物及探针，人工合成该区域序列并整合进质粒中作为检测标准品。对退火温度、引物浓度和探针浓度进行反应条件优化，建立TTV qPCR检测的标准曲线。收集武汉大学移植医学中心108例肾移植受者和等待肾移植患者临床血液样本，根据临床状态分为稳定组、感染组、排斥组和等待移植组，进行TTV载量检测，并通过受试者操作特征(ROC)曲线评估TTV拷贝数对肾移植术后感染和排斥反应的诊断价值。

最终确定qPCR优化条件为退火温度56 ℃，引物和探针加入量均为1 μL。根据优化后的qPCR反应条件，以1×10⁹~1×10³ copies/μL的标准品为模板进行qPCR检测生成标准曲线，决定系数为0.999，斜率为－3.320，截距为42.28，扩增效率为100.1%，最低可检测1×10¹ copies/μL病毒载量的样品。通过对108例患者血浆样本进行优化条件下的qPCR检测发现，TTV载量与感染或排斥反应的发生相关。稳定组受者TTV载量为5.125(4.550，5.491) log copies/μL，感染组TTV载量升高，为5.888(5.135，6.506) log copies/μL；排斥组TTV载量降低，为4.167(3.459~4.869)log copies/μL；移植前组TTV载量最低，为2.885(2.636，3.233) log copies/μL，差异均有统计学意义(P<0.05)。TTV载量诊断排斥反应的ROC曲线下面积为0.7875(P<0.05)，约登指数最大时截断值为4.999 log copies/μL；TTV载量诊断感染的ROC曲线下面积为0.7458(P<0.05)，约登指数最大时截断值为5.770 log copies/μL。

本研究构建的TTV qPCR检测具有良好的敏感度和特异度，可以用于人全血TTV的检测。当TTV载量低于9.98×10⁴ copies/μL时，发生排斥反应的风险较大；当TTV载量高于5.89×10⁵ copies/μL时，发生感染的风险较大。

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.