Rapid extraction-free detection of the R132H isocitrate dehydrogenase mutation in glioma using colorimetric peptide nucleic acid-loop mediated isothermal amplification (CPNA-LAMP).

Autor: Choate KA; Department of Biology, Northern Michigan University, Marquette, Michigan, United States of America.; Upper Michigan Brain Tumor Center, Marquette, Michigan, United States of America.; Northern Michigan University, Marquette, Michigan, United States of America., Raack EJ; Upper Michigan Brain Tumor Center, Marquette, Michigan, United States of America.; Northern Michigan University, Marquette, Michigan, United States of America.; School of Clinical Sciences, Northern Michigan University, Marquette, Michigan, United States of America., Line VF; Department of Biology, Northern Michigan University, Marquette, Michigan, United States of America.; Upper Michigan Brain Tumor Center, Marquette, Michigan, United States of America.; Northern Michigan University, Marquette, Michigan, United States of America., Jennings MJ; Upper Michigan Brain Tumor Center, Marquette, Michigan, United States of America.; Northern Michigan University, Marquette, Michigan, United States of America.; School of Clinical Sciences, Northern Michigan University, Marquette, Michigan, United States of America., Belton RJ Jr; Department of Biology, Northern Michigan University, Marquette, Michigan, United States of America.; Northern Michigan University, Marquette, Michigan, United States of America., Winn RJ; Department of Biology, Northern Michigan University, Marquette, Michigan, United States of America.; Upper Michigan Brain Tumor Center, Marquette, Michigan, United States of America.; Northern Michigan University, Marquette, Michigan, United States of America., Mann PB; Upper Michigan Brain Tumor Center, Marquette, Michigan, United States of America.; Northern Michigan University, Marquette, Michigan, United States of America.; School of Clinical Sciences, Northern Michigan University, Marquette, Michigan, United States of America.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2023 Sep 21; Vol. 18 (9), pp. e0291666. Date of Electronic Publication: 2023 Sep 21 (Print Publication: 2023).
DOI: 10.1371/journal.pone.0291666
Abstrakt: The R132H isocitrate dehydrogenase one (IDH1) mutation is a prognostic biomarker present in a subset of gliomas and is associated with heightened survival when paired with aggressive surgical resection. In this study, we establish proof-of-principle for rapid colorimetric detection of the IDH1-R132H mutation in tumor samples in under 1 hour without the need for a nucleic acid extraction. Colorimetric peptide nucleic acid loop-mediated isothermal amplification (CPNA-LAMP) utilizes 4 conventional LAMP primers, a blocking PNA probe complementary to the wild-type sequence, and a self-annealing loop primer complementary to the single nucleotide variant to only amplify the DNA sequence containing the mutation. This assay was evaluated using IDH1-WT or IDH1-R132H mutant synthetic DNA, wild-type or IDH1-R132H mutant U87MG cell lysates, and tumor lysates from archived patient samples in which the IDH1 status was previously determined using immunohistochemistry (IHC). Reactions were performed using a hot water bath and visually interpreted as positive by a pink-to-yellow color change. Results were subsequently verified using agarose gel electrophoresis. CPNA-LAMP successfully detected the R132H single nucleotide variant, and results from tumor lysates yielded 100% concordance with IHC results, including instances when the single nucleotide variant was limited to a portion of the tumor. Importantly, when testing the tumor lysates, there were no false positive or false negative results.
Competing Interests: The authors have declared that no competing interets exist.
(Copyright: © 2023 Choate et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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