Methodological Research on Rapid Detection and Genotyping of NPM1 Gene Mutations in Leukemia

Abstract

To establish a method for rapid detection and typing of NPM1 mutations in acute myeloid leukemia (AML) by fluorescence melting curve analysis technology. A pair of primers and a fluorescent single-stranded probe (molecule beacon) were designed for the mutant genes mutA, mutB, mutD in exon 12 of nucleopsin (NPM1) and wild type. With a real-time qPCR, the A, B, and D gene mutations of NPM1 were detected and typed by different-melting curve peak value of the probe through RT-PCR. This method could detected the mutations of A, B, and D in NPM1 effectively with a sensitivity of 1%. Furthermore, 62 AML clinical samples were evaluated by the method. In the results, the detection rate and typing of NPM1 mutations were consistent with the sequencing results of clinical samples. There are three features in the method of fluorescence melting curve analysis: stable PCR system, easy to operate, and the easily distinguishable results. The method might meet the demand for rapid typing of NPM1 gene mutation in early diagnosis or concomitant diagnosis of AML.