Quantification of T-cell receptor excision circle DNA using fluorescence resonance energy transfer and the LightCycler system
Quantification of T-cell receptor excision circle DNA using fluorescence resonance energy transfer and the LightCycler system
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DOI：
10.1016/s0022-1759(02)00337-x
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发表时间：
2002-12-20
影响因子：
2.2
通讯作者：
Einsele, H
中科院分区：
文献类型：
--
作者：
Loeffler, J;Bauer, R;Einsele, H
T细胞受体切除环（TRECs）是环状的、稳定的染色体外DNA片段，并且在V（D）J基因重组过程中产生，这是一个负责T细胞受体库多样性的过程。本文介绍了一种基于LightCycler技术的实时荧光定量PCR检测外周血细胞中TRECs的方法，并与传统的PCR-ELISA进行了比较，结果表明，LightCycler的检测灵敏度为20拷贝，而PCR-ELISA的检测限为2拷贝。在健康个体（n = 52）的血液样本中，TREC计数中位数为1.6 x 10（4）拷贝[范围为2 x 10（1）至2 x 10（5）]/2 x 10（5）外周血单核细胞（PBMNC），可检测到中位数TREC计数为1.45 × 10（5）拷贝[范围为1.2 × 10（5）至1.6 × 10（5）]/2 × 10（5）PBMC。当分析未分级PBMC（中位数计数为1.1 x 10（5）拷贝/2 x 10（5）PBMC）或磁相关细胞分选仪（MACS）分选的CD 45 RA + T细胞（中位数计数为2.5 x 10（5）拷贝/2 x 10（5）细胞）的TREC计数时，在15名个体中未发现显著差异。此外，我们检查了异基因干细胞移植后儿童（n = 6）和成人患者（n = 7）中deltaRec-psiJa信号关节TREC的数量。在儿童中，我们观察到TREC计数中位数为5.7 x 10（4）拷贝/2 x 10（5）PBMNCs，1 x 10（4）和5.6 x 10（4）拷贝/2 x 10（5）PBMNCs后2年，在成人中，1 x 10（4）年后的中位计数为3.6 x 10（4）拷贝/2 x 10（5）PBMNC，2年后为1.1 x 10（4）拷贝/2 x 10（5）PBMNC。总之，所描述的基于LightCycler的实时PCR测定为TREC DNA的定量提供了非常灵敏和快速的工具。(C)2002 Elsevier Science B. V.保留所有权利。
T-cell receptor excision circles (TRECs) are circular, stable extrachromosomal DNA fragments and are generated during V(D)J gene recombination, a process responsible for diversity of the T-cell receptor repertoire. Here we describe a sensitive, rapid and easy to perform real-time PCR assay based on the LightCycler technique for the quantification of TRECs among peripheral blood cells and a comparison of this assay with conventional PCR-ELISA.By LightCycler, a sensitivity of 20 copies of plasmid deltaRec-psiJa Signal Joint TREC DNA was achieved whereas by PCR-ELISA, a detection limit of 2 copies was demonstrated. In blood samples from healthy individuals (n = 52) a median TREC count of 1.6 x 10(4) copies [range 2 x 10(1) to 2 x 10(5)]/2 x 10(5) peripheral blood mononuclear cells (PBMNCs) and in cord blood, a median TREC count of 1.45 x 10(5) copies [range 1.2 x 10(5) to 1.6 x 10(5)]/2 x 10(5) PBMNCs) could be detected. No significant difference was found in 15 individuals when unfractionated PBMNCs (median count of 1.1 x 10(5) copies/2 x 10(5) PBMNCs) or magnetic associated cell sorter (MACS)-sorted CD45RA+ T-cells (median count of 2.5 x 10(5) copies/2 x 10(5) cells) were analyzed for TREC counts. In addition, we examined the number of deltaRec-psiJa Signal Joint TREC in paediatric (n = 6) and adult patients (n = 7) after allogeneic stem cell transplantation. In children, we observed a median TREC count of 5.7 x 10(4) copies/2 x 10(5) PBMNCs after 1 x 10(4) and 5.6 x 10(4) copies/2 x 10(5) PBMNCs after 2 years, and in adults, a median count of 3.6 x 10(4) copies/2 x 10(5) PBMNCs after 1 x 10(4) and 1.1 x 10(4) copies/2 x 10(5) PBMNCs after 2 years. In conclusion, the LightCycler-based real-time PCR assay described offers a very sensitive and rapid tool for the quantification of TREC DNA. (C) 2002 Elsevier Science B.V. All rights reserved.