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目的:建立BCR/ABL融合基因表达定量分析的双色荧光定量PCR技术;探讨所建立的双色荧光定量PCR技术在CML早期诊断中的临床应用特点。方法:据BCR/ABL融合基因序列,设计合成BCR/ABL融合基因及ABL基因特异的引物和TaqMan探针。提取患者骨髓细胞标本总RNA,逆转录成cDNA;在同一PCR反应管中同时进行两种基因的双色荧光定量PCR反应,分别定量测定BCR/ABL融合基因和ABL基因的表达量,并计算两种基因表达的比值。检测标本中两种基因的表达量,计算检测的阳性率,所得结果与临床诊断结果对比,以判断该技术在CML检测诊断中的价值。结果:成功地建立了BCR/ABL融合基因检测的双色荧光定量PCR技术方法;在35例临床CML患者中,33例检出融合基因阳性,检测阳性率94.3%;12例正常人cDNA均未检出融合基因阳性。结论:所建立的BCR/ABL融合基因检测的双色荧光定量PCR是一种快速、准确、高通量而费用又较为低廉的基因检测技术。
OBJECTIVE: To establish a two-color fluorescence quantitative PCR technique for the quantitative analysis of BCR / ABL fusion gene expression and to explore the clinical application of the established two-color fluorescence quantitative PCR technique in the early diagnosis of CML. Methods: According to the sequence of BCR / ABL fusion gene, BCR / ABL fusion gene and ABL gene specific primer and TaqMan probe were designed and synthesized. The total RNA was extracted from the bone marrow samples of patients and reverse transcribed into cDNA. Two-color fluorescence quantitative PCR reaction was carried out simultaneously in the same PCR reaction tube to quantify the expression of BCR / ABL fusion gene and ABL gene, The ratio of gene expression. Detecting the expression of two genes in the specimen and calculating the positive rate of the test. The obtained results are compared with the clinical diagnosis results to determine the value of the technique in the diagnosis of CML. Results: Two-color fluorescence quantitative PCR was successfully established for BCR / ABL fusion gene detection. Among the 35 CML patients, the positive rate of fusion gene was detected in 33 cases, and the positive rate was 94.3%. All 12 normal human cDNA samples were undetected The fusion gene positive. Conclusion: The established two-color fluorescent quantitative PCR for BCR / ABL fusion gene detection is a fast, accurate, high-throughput and low-cost genetic testing technique.