Real-time reverse transcription loop-mediated isothermal amplification for rapid and simple quantification of WT1 mRNA

ObjectivesThis study developed a novel MRD monitoring method targeting Wilms' tumor gene (WT1) mRNA using reverse transcription loop-mediated isothermal amplification (RT-LAMP).Design and methodsA primer set for the assay was designed on the basis of the sequences between the 17AA and KTS regions of WT1 mRNA. WT1 mRNA was quantified by real-time RT-LAMP and the accuracy of RT-LAMP was compared with that of real-time RT-PCR.ResultsThe standard curve was expressed as a linear relationship between the log copy numbers of WT1 mRNA ranging from 6.8 × 10 to 6.8 × 109 copies and the threshold time with a correlation coefficient of R2 > 0.994. The measured values obtained by RT-LAMP strongly correlated with those obtained by real-time RT-PCR.ConclusionRT-LAMP can be used to determine WT1 mRNA expression levels. This assay will contribute to a more specific, simple, and rapid MRD monitoring than conventional assays.