Ultra-fast detection and quantification of nucleic acids by amplification-free fluorescence assay

Jesper Uhd, Laura Miotke, Hanlee P Ji, Marina Dunaeva, Ger J M Pruijn, Christian Damsgaard Jørgensen, Emil Laust Kristoffersen, Victoria Birkedal, Christina Westmose Yde, Finn Cilius Nielsen, Jonas Hansen, Kira Astakhova

Abstract

Two types of clinically important nucleic acid biomarkers, microRNA (miRNA) and circulating tumor DNA (ctDNA) were detected and quantified from human serum using an amplification-free fluorescence hybridization assay. Specifically, miRNAs hsa-miR-223-3p and hsa-miR-486-5p with relevance for rheumatoid arthritis and cancer related mutations BRAF and KRAS of ctDNA were directly measured. The required oligonucleotide probes for the assay were rationally designed and synthesized through a novel "clickable" approach which is time and cost-effective. With no need for isolating nucleic acid components from serum, the fluoresence-based assay took only 1 hour. Detection and absolute quantification of targets was successfully achieved despite their notoriously low abundance, with a precision down to individual nucleotides. Obtained miRNA and ctDNA amounts showed overall a good correlation with current techniques. With appropriate probes, our novel assay and signal boosting approach could become a useful tool for point-of-care measuring other low abundance nucleic acid biomarkers.

OriginalsprogEngelsk
TidsskriftThe Analyst
Vol/bind145
Udgave nummer17
Sider (fra-til)5836-5844
Antal sider9
ISSN0003-2654
DOI
StatusUdgivet - 24 aug. 2020

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