Establishing The AS-PCR Method For Mutation NPM1Detection And Its Application Of Leukemia

Objective：To establish an allele-specific PCR（AS-PCR）method to detect the mutation ofNPM1gene, provide a sensitive, reliable and simple method for clinical detectionof NPM1gene mutations in acute myeloid leukemia patients.Methods：1. The site-directed mutation cloning primers and wild type cloning primers forNPM1gene were designed and used to amplified the mutant and wild type NPM1gene, by using the genomic DNA of healthy human peripheral blood as template. ThePCR products were then inserted into the pEGFP-C1plasmid. The recombinantplasmids were used as the positive and negative control for detection of NPM1genemutation.2. A pair of primers were designed according the sequence of NPM1gene andthe principle of allele-specific PCR （AS-PCR）. The annealing temperature wasoptimized by using recombinant plasmids containing mutant NRM1gene fragment asPCR template. The sensitivity, specificity, and interference test method to conduct afeasibility analysis.3The established AS-PCR method was used to detect the mutation of NPM1gene in acute leukemia patients. The relation between NPM1mutation and abnormalFAB classification or genetics was evaluated.Results：1. Recombinant plasmids containing wild NPM1or mutant NPM1gene wereconstructed sucessfully.2. The detection limit of the established AS-PCR were10³copies/ml to10⁹copies/ml. Wild-type NPM1gene had no interference to the detection of mutant NPM1gene when the concentration lower than10⁹copies/ml.3.231leukemia patients were selected to detect the mutation of NPM1gene byAS-PCR. The mutation of NPM1was detected in sixty-two patients. The mutation rate of NPM1was26.8%in all acute leukemia patients, mainly in the M₅and M₂typeleukemia.Conclusion：The positive and negative control plasmids were constructed rightly, establishedan AS-PCR method to detecting the mutation of NPM1gene, it is a sensitive, reliableand simple method for detecting the mutation of NPM1gene.