论文部分内容阅读
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。
OBJECTIVE: To establish a real-time quantitative real-time PCR method for quantitative simultaneous detection of multiple leukemia chromosomal translocations to understand the feasibility of array PCR. Methods Using 82 primers in combination, 66 PCR parallel tubes were set up, and 125 kinds of splices formed by 37 common fusion genes of leukemia and 4 kinds of protooncogenes common in leukemia were detected quantitatively. Real-time quantitative PCR using Eva Green fluorescent dye method, with the ABL gene as a reference, comparative Ct method for relative quantification. Thirty-one patients with leukemia were detected by this protocol. Six patients with chronic myeloid leukemia (CML) were tested before and after treatment or at different times during treatment, and 28 were compared with the results of multiple nested PCR. Results The PCR array protocol we developed had a large linear detection range (10 ~ 2 ~ 10 ~ 8 copies / μl) with high detection sensitivity (232 copies / μl) and accuracy. The detection of 31 patients , A total of 14 fusion genotypes and all four oncogenes were detected. One patient with a positive fusion gene and two active oncogenes was detected; and a comparison of multiplex nested PCR results showed that The sensitivity of the protocol was slightly lower than that of nested PCR, but the difference was not statistically significant (P = 0.009). The detection of 6 specimens of CML patients showed that the expression of BCR / ABL fusion gene and WT1 and EVI1 oncogenes were all The results of gene quantitative analysis showed that this program can quantitatively analyze the common leukemia fusion gene and oncogene activation, and has the value of application in the diagnosis and curative effect monitoring of leukemia. Conclusions The PCR array method is also used to detect the fusion genes of many leukemia fusion genes in newly diagnosed leukemia patients and the detection of minimal residual disease (MRD). Simultaneous detection of multiple fusion genes and the fusion genes during treatment Variation is more useful.