ARUP Institute for Clinical and Experimental Pathology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT.
Clin Chem. 2020 Jan 1;66(1):229-238. doi: 10.1373/clinchem.2019.308171.
T-cell receptor excision circles (TREC) and κ-deleting recombination receptor excision circles (KREC) concentrations can be used to assess and diagnose immune deficiencies, monitor thymic and bone marrow immune reconstitution, or follow responses to drug therapy. We developed an assay to quantify TREC, KREC, and a reference gene in a single reaction using droplet digital PCR (ddPCR).
PCR was optimized for 3 targets: TREC, KREC, and ribonuclease P/MRP subunit p30 (RPP30) as the reference gene. Multiplexing was accomplished by varying the target's fluorophore and concentration. Correlation with clinical results was evaluated using 47 samples from healthy donors, 59 samples with T-cell and B-cell markers within the reference interval from the flow cytometry laboratory, 20 cord blood samples, and 34 samples submitted for exome sequencing for severe combined immunodeficiency disease (SCID).
The limit of the blank was 4 positive droplets, limit of detection 9 positive droplets, and limit of quantification 25 positive droplets, or 2.0 copies/μL. TREC and KREC copies/μL were as expected in the healthy donors and cord blood samples and concordant with the healthy flow cytometry results. Of the samples from the SCID Panel, 56.5% had a TREC count <20 copies/μL and 17.7% had a KREC count <20 copies/μL, suggestive of low T- and B-cell numbers, respectively.
Our multiplex ddPCR assay is an analytically sensitive and specific method for the absolute quantification of TREC and KREC. To the best of our knowledge, this paper is the first to describe the simultaneous quantification of TREC, KREC, and a reference gene by use of ddPCR.
T 细胞受体切除环（TREC）和 κ 缺失重组受体切除环（KREC）浓度可用于评估和诊断免疫缺陷，监测胸腺和骨髓免疫重建，或随访药物治疗反应。我们开发了一种使用液滴数字 PCR（ddPCR）在单个反应中定量 TREC、KREC 和参比基因的检测方法。
针对 3 个靶标（TREC、KREC 和核糖核酸酶 P/MRP 亚基 p30（RPP30）作为参比基因）优化了 PCR。通过改变靶标的荧光基团和浓度来实现多重检测。通过来自健康供体的 47 个样本、流式细胞仪实验室中 T 细胞和 B 细胞标志物在参考区间内的 59 个样本、20 个脐带血样本和 34 个提交用于严重联合免疫缺陷病（SCID）外显子测序的样本来评估与临床结果的相关性。
空白的检测限为 4 个阳性液滴，检测限为 9 个阳性液滴，定量限为 25 个阳性液滴，或 2.0 拷贝/μL。健康供体和脐带血样本中的 TREC 和 KREC 拷贝/μL 与预期相符，且与健康流式细胞术结果一致。在 SCID 面板样本中，56.5%的 TREC 计数<20 拷贝/μL，17.7%的 KREC 计数<20 拷贝/μL，分别提示 T 细胞和 B 细胞数量较低。
我们的多重 ddPCR 检测方法是一种分析敏感和特异的方法，用于绝对定量 TREC 和 KREC。据我们所知，本文首次描述了使用 ddPCR 同时定量 TREC、KREC 和参比基因。
