Development and application of a highly sensitive method for detection of mutation of FMS-like tyrosine kinase-3-tyrosine kinase domain of acute myeloid leukemia
10.13200/j.cnki.cjb.003552
- VernacularTitle:急性髓系白血病FMS样酪氨酸激酶3-激酶结构域突变的高灵敏度检测方法的建立及应用
- Author:
ZHUANG Xiaomei
- Publication Type:Journal Article
- Keywords:
Acute myeloid leukemia(AML);
FMS-like tyrosine kinase-3(FLT3)-tyrosine kinase domain(TKD);
Sensitivity;
Minimal residual disease(MRD)
- From:
Chinese Journal of Biologicals
2023;36(3):330-
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo develop a highly sensitive method for detection of mutation of FMS-like tyrosine kinase-3-tyrosine kinase domain(FLT3-TKD)of acute myeloid leukemia(AML)and apply to the monitor of minimal residual disease(MRD).MethodsRecombinant plasmids containing wild FLT3 and mutant FLT3-D835Y were constructed respectively and mixed at certain ratios.The obtained standard plasmids with mutation rates of 50%,1%,0.1% and 0% respectively were determined by restriction fragment length polymorphism(RFLP)in combination with Sanger method.The plasmid DNA standards and blood DNA standards,at various FLT3-D835Y mutation rates,were determined by the developed method to verify the sensitivity.The genomic DNA samples of patients with AML before and after treatment were determined by the developed method to monitor the MRD.ResultsSequencing proved that both the recombinant plasmids containing wild FLT3 and mutant FLT3-D835Y were constructed correctly.The sensitivity of developed method increased to 0.1% through Sanger method combined with digestion with EcoR Ⅴ/Xho Ⅰ and recovery of mutant fragments in determination of purified plasmid DNA and collected blood DNA samples.MRD was detected in the peripheral blood sample of a patients with AML in complete remission period by the developed method but not by Sanger method.ConclusionA highly sensitive method for detection of FLT3-TKD mutation was developed,which was of an important clinical significance in guiding the treatment of AML and monitoring the MRD in complete remission period.
