Rapid detection of clonal T-cell receptor-β gene rearrangements in T-cell lymphomas using the LightCycler-polymerase chain reaction with DNA melting curve analysis
Rapid detection of clonal T-cell receptor-β gene rearrangements in T-cell lymphomas using the LightCycler-polymerase chain reaction with DNA melting curve analysis
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DOI：
10.1016/s1525-1578(10)60012-8
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发表时间：
2005-02-01
影响因子：
4.1
通讯作者：
Ratech, H
中科院分区：
文献类型：
--
作者：
Yang, XY;Xu, DS;Ratech, H
已经开发了各种分子方法来诊断临床样品中的克隆T细胞受体（TCR）基因重排。大多数用于检测克隆TCR基因重排的聚合酶链反应策略依赖于凝胶或毛细管电泳。然而，繁琐的手动转移步骤将扩增与分析分开。最近，我们开发了一种新的聚合酶链反应检测使用LightCycler系统检测克隆免疫球蛋白重链基因重排。在目前的研究中，我们将这项工作扩展到包括TCR。我们报告说，克隆。通过在单个封闭毛细管中扩增后立即测量DNA熔解，可以在制备DNA后不到1小时内检测到TCR-β（TCR-β）基因重排。我们回顾性研究了52例临床疑似T细胞恶性肿瘤患者的新鲜冷冻组织样本。DNA熔解曲线分析发现14例标本存在TCR β基因重排。当将DNA熔解与Southern印迹或变性梯度凝胶电泳的金标准方法进行比较时，其达到了等于71%的灵敏度和等于94%的特异性。我们还比较了熔解曲线分析和聚丙烯酰胺凝胶电泳：熔解曲线分析的敏感性等于100%，特异性等于97%。我们的结论是，在LightCycler系统中的DNA熔解曲线分析作为一种新的，超快速的方法，初步诊断克隆TCR-β基因重排的临床应用潜力。
Various molecular methods have been developed to diagnose clonal T-cell receptor (TCR) gene rearrangements in clinical samples. Most polymerase chain reaction strategies for detecting clonal TCR gene rearrangements rely on either gel or capillary electrophoresis. However, a cumbersome manual transfer Step separates amplification from analysis. Recently, we developed a novel polymerase chain reaction assay using the LightCycler system to detect clonal immunoglobulin heavy chain gene rearrangement. In the current study, we extend this work to include the TCR. We report that clonal. TCR-beta (TCR-beta) gene rearrangements can be detected in less than I hour after preparing the DNA by measuring DNA melting immediately after amplification in a single closed capillary tube. We retrospectively studied 52 fresh-frozen tissue samples from patients clinically suspected of T-cell malignancy. A clonal TCR-beta gene rearrangement was detected in 14 samples by DNA melting curve analysis. When DNA melting was compared to the gold standard methods of Southern blot or denaturing gradient gel electrophoresis, it achieved a sensitivity equal to 71% and a specificity equal to 94%. We also compared melting curve analysis and polyacrylamide gel electrophoresis: melting curve analysis reached a sensitivity equal to 100% and a specificity equal to 97%. We conclude that DNA melting curve analysis in the LightCycler system has potential for clinical use as a new, ultra-fast method for the initial diagnosis of clonal TCR-beta gene rearrangements.