[Simultaneous detection of FLT3-ITD and NPM1 gene mutations in acute myeloid leukemia by double PCR].

Objective of this study was to establish a method for simultaneous detection of FLT3/ITD and NPM1 gene mutations in AML.A double PCR was firstly designed and optimized to amplify both exon 12 of NPM1 and exon 14-intron 14-exon 15 of FLT3,with the aim of detecting almost all reported mutations.After optimization,a touchdown PCR was chosen for the multiplex PCR procedure,with the primer concentrations of NPM1 and FLT3-ITD being 200 nmol/L and 152 nmol/L respectively.The PCR amplicons were separated by capillary electrophoresis and the presence of mutants was recognized by the size difference between the mutants and wild-type products.The areas of mutant peak and wild-type peak were used to calculate the mutant/wild-type ratio.All the positive mutated samples were confirmed by sequencing.The results showed that 17 patients with NPM1 mutation,15 patients with FLT3-ITD mutation,6 patients with both NPM1 and FLT3-ITD mutationts were found among 93 patents.7 patients with M2,4 patients with M4,5 patients with M5 and 1 patients with M6 were found out of 17 patients with NPM1 mutation,in which 10 patients were male and 7 patients were female,15 patients were with type A,1 patients was with type B and 1 patients was with type Nm,strinkingly 1 CML patient in blast crisis was found to carry a type A mutation.Among 15 patients with FLT3-ITD mutation 1 patient with M1,8 patients with M2,2 patients with M2,2 patients with M3,1 patient with M4,3 patients with M5 were found,in which 5 patients were male and 10 patients were lemale.Sequencing results further confirmed the accuracy and reliability of this method.It is concluded that a novel method with the ability to detect both FLT3-ITD and NPM1 mutations has been developed when genomic DNA was templated.This method is fast,easy,accurate and capable to calculate the mutant/wild-type ratio.