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目的用改良的杂交探针结合溶解曲线新方法检测急性髓性白血病c-kit基因17号外显子上基因突变。方法利用Primer Premierv5.00、Tm Utilityv1.3等软件设计引物探针,两个阶段探针分别针对D816、N820和N822等突变热点。取患者DNA样品5μl进行PCR扩增及熔解曲线分析,PCR扩增产物进行测序分析并与熔解曲线分析方法进行比较。结果阳性质粒灵敏度试验的结果显示,N820G的灵敏度为10%,其余突变灵敏度均能达到5%。12例CBF-AML标本中,5例检测为阳性,突变率为41.7%,测序结果与测序检测结果相符。结论新方法具有方便快速、灵敏度高、特异性好等特点,可用于CBF-AML患者的个体化诊断和治疗。
OBJECTIVE: To detect the mutation of exon 17 in c-kit gene of acute myeloid leukemia by a new hybridization probe and a new dissolution curve method. Methods The Primer Premierv5.00 and Tm Utilityv1.3 softwares were used to design primer probes. The two-step probes were targeted to mutational hot spots such as D816, N820 and N822. Take 5μl of the patient’s DNA sample for PCR amplification and melting curve analysis. The PCR amplification product was sequenced and compared with the melting curve analysis method. Results The results of the positive plasmid sensitivity test showed that the sensitivity of N820G was 10% and the sensitivity of other mutants could reach 5%. Of the 12 CBF-AML specimens, 5 were positive, with a mutation rate of 41.7%. The sequencing results were in good agreement with the sequencing results. Conclusion The new method is convenient, rapid, sensitive and specific. It can be used in the individual diagnosis and treatment of CBF-AML patients.