A RAPID COLORIMETRIC PEPTIDE NUCLEIC ACID LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD FOR THE DETECTION OF THE IDH1 MUTATION IN GLIOBLASTOMA

The IDH1 mutation is an important diagnostic and prognostic biomarker used to characterize glioblastoma (GBM). Patients harboring the IDH1 mutation have improved overall survival following maximal resection. Knowledge of the IDH1 mutation status allows the surgeon to modify the surgical plan; however, no existing molecular test can provide this information intraoperatively. We designed a novel colorimetric peptide nucleic acid loop-mediated isothermal amplification (PNA-LAMP) method that rapidly detects the IDH1 R132H mutation in GBM. PNA-LAMP amplifies target DNA under isothermal conditions with high specificity and speed. The PNA prevents amplification of wild-type IDH1 DNA, while amplifying the R132H variant if present. We used a pH-sensitive colorimetric detection method to allow visual determination of amplification, eliminating the need for specialized instrumentation. Initial characterization of the assay was performed with plasmid DNA containing the wild-type and R132H variant sequences. Initially, we were able to amplify exon four of the IDH1 gene in both genomic DNA and whole cell lysates using the conventional LAMP method. However, when LAMP was applied to plasmid DNA containing the R132H variant, it could not differentiate between wild-type and mutant. The inclusion of the PNA prevented amplification of wild-type while allowing amplification of the R132H variant. This assay allowed for the visual detection of the mutant sequence in under one hour. Amplification was confirmed using agarose electrophoresis, and this analysis suggests that sensitivity of the visual detection method can be improved up to six-fold. This novel molecular assay is a simple, specific, and rapid way to identify the presence of the IDH1 R132H variant associated with GBM. Our current efforts are focused to develop this assay for intraoperative diagnostics.