聚合酶链反应阵列同时定量检测37种白血病融合基因

目的建立一种同时定量检测多种白血病染色体易位形成的融合基因的荧光实时定量聚合酶链反应（real-time PCR）方法,了解阵列式PCR的应用可行性。方法组合使用82条引物,设立66个PCR平行管,同时定量检测37种白血病常见融合基因形成的125种剪切体和4种白血病常见的原癌基因活化。实时定量PCR采用Eva Green荧光染料法,用ABL基因做内参,用比较Ct法进行相对定量。用此方案对31例白血病患者标本进行检测,其中6例慢性粒细胞白血病（CML）患者做治疗前后或治疗过程中不同时间的对比检测,28人次与多重巢式PCR方案的检测结果进行对照。结果我们建立的PCR阵列方案有较大的线性检测范围（10^2～10^8拷贝／μl）,有较高的检测灵敏度（232拷贝／μl）和精确性;在31例患者标本的检测中,共检测到14种融合基因类型和所有4种原癌基因活化;检测到一例同时有5种融合基因阳性和两种原癌基因活化的患者;和多重巢式PCR检测结果的对比显示本方案灵敏度略低于巢式PCR,但差异没有统计学意义（P=0．009）;6例CML患者标本的检测显示治疗后患者标本中BCR／ABL融合基因及WT1和EVI1原癌基因表达量均呈现不同程度的降低,与临床治疗情况符合;基因定量分析的结果表明本方案能够对常见白血病融合基因和癌基因活化情况进行定量分析,在白血病的诊断及疗效监测中有应用价值。结论PCR阵列法同时定量检测多种白血病融合基因适于白血病初诊患者融合基因的筛查及微小残留病（MRD）的检测,对于检测同时有多种融合基因存在的病例及治疗过程中融合基因的变异情况更为有用。

Real-time PCR array for simultaneous detection of 37 kinds of fusion genes in leukemia

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.