论文部分内容阅读
目的建立高分辨熔解曲线分析(HRMA)技术检测急性髓细胞白血病(AML)中可溶性异柠檬酸脱氢酶2(IDH2)基因突变。方法针对IDH2突变位点设计PCR扩增引物,建立带有温度内标校准品的PCR扩增体系,对98例AML患者标本进行PCR扩增,用HRMA技术对PCR产物进行突变分析,并通过DNA测序技术对HRMA结果进行验证。结果对98例AML样本的PCR产物进行HRMA检测,发现5例存在异常熔解曲线,对此5例标本及随机选择的HRMA显示为野生型的35例IDH2标本进行DNA测序验证,结果表明,存在异常熔解曲线的5例标本为4例R140Q突变和1例R172K突变,其余35例经DNA测序证实为野生型IDH2;对不同浓度梯度的R140Q质粒HRMA检测结果表明其灵敏度为2%。结论成功建立HRMA技术检测IDH2突变,其可用于AML标本的临床检测。
Objective To establish a high resolution melting curve analysis (HRMA) technique for the detection of soluble isocitrate dehydrogenase 2 (IDH2) gene mutations in acute myeloid leukemia (AML). Methods PCR amplification primers were designed according to the IDH2 mutation site. A PCR amplification system with temperature internal standard calibration was established. 98 cases of AML patients were amplified by PCR. The PCR products were analyzed by HRMA and analyzed by DNA Sequencing techniques validate HRMA results. Results The PCR products of 98 cases of AML samples were detected by HRMA, and 5 cases were found to have abnormal melting curve. DNA sequencing of 35 cases and IDH2 samples of wild type randomly selected HRMA showed that there were abnormalities Five samples of the melting curve were four cases of R140Q mutation and one case of R172K mutation, and the remaining 35 cases were confirmed as wild-type IDH2 by DNA sequencing. The HRMA results of R140Q plasmid with different concentration gradients showed that the sensitivity was 2%. Conclusion HRMA technique was successfully established to detect IDH2 mutation, which can be used for the clinical detection of AML specimens.