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目的:建立一种应用变性高效液相色谱技术(DHPLC)相对定量检测急性髓细胞白血病(AML)患者Fms样酪氨酸激酶3(FLT3)基因的内部串联重复(ITD)突变的方法。方法:根据FLT3-ITD突变基因多位于14外显子而设计引物,用聚合酶链反应(PCR)方法特异性扩增121例AML患者FLT3-ITD突变基因,再用DH-PLC技术相对定量检测FLT3-ITD等位基因突变的情况;与毛细管电泳法(CE)检测突变的结果对比进行该方法的有效性检验;最后与121例样品PCR扩增产物的测序结果进行对比。结果:经DHPLC分析后均能得到特征性的洗脱峰。121例样本中检测到FLT3-ITD突变阳性的样本13例,总阳性率为10.7%,阳性突变等位基因的比例不一,分布范围中位数为34.5%(11.4%-80.2%),为21-87 bp单个插入片段。阳性率和突变比例与CE方法检测结果相比较均无显著差异(P>0.05),并与121例样本FLT3-ITD扩增PCR产物基因测序结果一致。结论:成功建立了一种应用DHPLC相对定量检测AML患者FLT3-ITD基因突变的方法。
OBJECTIVE: To establish a method for the relative quantitation of internal tandem repeat (ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) gene in patients with acute myeloid leukemia (AML) by denaturing high performance liquid chromatography (DHPLC) Methods: According to the FLT3-ITD mutant gene located in exon 14, primers were designed to amplify the FLT3-ITD mutation gene in 121 cases of AML by polymerase chain reaction (PCR) and then quantitatively detected by DH-PLC FLT3-ITD allele. The validity of this method was compared with that of capillary electrophoresis (CE). Finally, the sequencing results of 121 PCR products were compared. Results: The characteristic elution peaks were obtained after DHPLC analysis. Among 121 samples, 13 samples were positive for FLT3-ITD mutation, the total positive rate was 10.7%. The proportion of positive mutation alleles varied from 34.5% (11.4% -80.2%) to 21-87 bp single insert. There was no significant difference (P> 0.05) between the positive rate and mutation rate and CE test results, which was in good agreement with the results of FLT3-ITD PCR amplification of 121 samples. CONCLUSION: A method to detect FLT3-ITD gene mutation in AML patients by DHPLC was established successfully.