One-Pot Ligation LAMP Assay to Detect miRNA-222: A Glioma Biomarker

Abstract

Many cancer diagnostic methods are invasive, time-consuming, and expensive. Delayed cancer diagnosis can lower patient survival rates. PCR-based techniques that detect miRNA biomarkers in blood have been utilized as early screening tools for various cancers. As an alternative to PCR, we designed and optimized an isothermal amplification technique, Ligation Loop-Mediated Isothermal Amplification (Ligation LAMP) assay, to detect miR-222, an established biomarker that is found in elevated levels in the bloodstream of early-stage glioma patients. We designed colorimetric and fluorescent Ligation-LAMP assays and demonstrated their specificity and sensitivity in detecting miR-222. We are working on implementing our assay into a One-Pot system, using Thermally Responsive Alkane Partitions (TRAPs) and a strand displacement assay utilizing magnetic beads. We found that the Ligation LAMP assay is sensitive and specific to glioma biomarker miR-222 and different probe lengths for Strand Displacement did not have a significant impact on ligation. With these results, we can further improve the One-Pot assay to be more point-of-care.