Liu Hong-xing, Zhu Ping, Zhang Ying, Wang Hong-xi, DU Jin-wei, Liu Jing, Gu Jiang-ying, Ou Yuan
Daopei Hospital, Beijing 100034, China.
Zhonghua Yi Xue Za Zhi. 2007 Feb 27;87(8):526-32.
To develop a real-time PCR array for simultaneous quantitative detection of translocations/chromosomal aberrations in patients with leukemia, and to investigate the feasibility and utility thereof.
By construction and optimization a set of specific primes (totally 82 primers), an array containing 66 parallel PCR reactions was developed. That array was used on the specimens of bone marrow or peripheral blood from 31 patients with leukemia to detect simultaneously 37 fusion genes and 4 proto-oncogene activations often occurring in patients with leukemia. Eva Green fluorescent dye method was chosen in the protocol. Relative quantification was performed by Ct analysis and the result was expressed as the ratio of the target gene versus the internal control gene (ABL). Six patients with chronic myelocytic leukemia (CML) among the 31 cases underwent prior to and after treatment so as to study the expression changes of fusion genes and/or proto-oncogene.
The established PCR array showed high efficiency of amplification and good sensibility (232 copies/microl) in the fusion gene detected. The standard curve had a satisfying linear range (10(2) approximately 10(8) copies/microl), showing a good reproducibility. Fourteen fusion genes, including PML/RARalpha, PLZF/RARalpha, BCR/ABL, MLL/AF1, MLL/AF6, MLL/AF10, AML/Eto, CBFbeta/MYH11, TLS/ERG, TEL/AML1, MOZ/CBP, MLL/hCDCrel, LAF4/MLLT2, and FIP1L1/PDGFRalpha, and activation of all 4 proto-oncogenes were found in the 31 samples. In one patient, 5 fusion genes and activation of 2 proto-oncogenes were observed. Such results were compared with those of RT-nested PCR in 28 samples. The comparison showed that this array was a bit less sensitive than RT-nested PCR, however, without significant difference between them (P = 0.009). The expression of BCR/ABL fusion gene, WT1 gene, and EVI1 gene decreased after treatment in the 6 CML patients, which was in accordance with the clinical features.
The PCR array newly-established successfully detects various leukemia related fusion genes and proto-oncogene activation. It is useful in molecule diagnosis and monitoring minimal residual disease in leukemia, and therapeutic effect monitoring.
研发一种用于同时定量检测白血病患者易位/染色体畸变的实时PCR芯片，并探讨其可行性和实用性。
通过构建和优化一组特异性引物（共82条引物），开发出一个包含66个平行PCR反应的芯片。该芯片用于检测31例白血病患者的骨髓或外周血标本，以同时检测白血病患者中常见的37种融合基因和4种原癌基因激活情况。实验方案选用Eva Green荧光染料法。通过Ct分析进行相对定量，结果以目标基因与内参基因（ABL）的比值表示。31例患者中有6例慢性粒细胞白血病（CML）患者在治疗前后进行检测，以研究融合基因和/或原癌基因的表达变化。
所建立的PCR芯片在检测融合基因时显示出高效扩增和良好的敏感性（232拷贝/微升）。标准曲线具有令人满意的线性范围（10²至10⁸拷贝/微升），显示出良好的重复性。在31个样本中发现了14种融合基因，包括PML/RARα、PLZF/RARα、BCR/ABL、MLL/AF1、MLL/AF6、MLL/AF10、AML/Eto、CBFβ/MYH11、TLS/ERG、TEL/AML1、MOZ/CBP、MLL/hCDCrel、LAF4/MLLT2和FIP1L1/PDGFRα，以及所有4种原癌基因的激活。在1例患者中，观察到5种融合基因和2种原癌基因的激活。将这些结果与28个样本的RT嵌套PCR结果进行比较。比较结果显示，该芯片的敏感性略低于RT嵌套PCR，但两者之间无显著差异（P = 0.009）。6例CML患者治疗后BCR/ABL融合基因、WT1基因和EVI1基因的表达下降，这与临床特征相符。
新建立的PCR芯片成功检测到多种白血病相关融合基因和原癌基因激活。它在白血病的分子诊断、微小残留病监测及治疗效果监测中具有重要应用价值。
