Colorimetric PCR-Based microRNA Detection Method Based on Small Organic Dye and Single Enzyme

Abstract

microRNAs (miRNAs) have been a class of promising disease diagnostic biomarkers and therapeutic targets for their important biological functions. However, because of the high homology, interference from precursors (pri-miRNA, pre-miRNA), as well as limitations in the current assay technologies, it poses high demand and challenge for a specific, efficient, and economic miRNA assay method. Here, we propose a new miRNA detection method based on a label-free probe and a small organic dye with sequence dependence, realizing the sequence-specific and colorimetric detection of target miRNA. What is pleasantly surprising, only one enzyme is enough to propel the whole miRNA assay process, greatly simplifying the reaction component and detection process. Together with PCR amplification for the high enough sensitivity and three checks for specificity control, a detection limit of 5 fM was obtained and even one mutation could be discriminated visually. Overall, the new method makes much progress in convenience and economy of PCR-based miRNA assay method so that miRNA assay is going to be more friendly and affordable.

Enhanced electrochemiluminescence of Ru(bpy)32+‐doped silica nanoparticles by chitosan/Nafion shell@carbon nanotube core‐modified electrode

“…In addition, as listed in Table 1, the sensitivity of the proposed biosensor was comparable with, or better than, that of other previously reported approaches for detection of microRNA. [ 21–26 ]…”

Nucleic Acids Analysis

“…While the early generations of PCR-based detection technology are timeconsuming and cannot be used to quantitatively detect target nucleic acids, especially RNA samples, quantitative reverse transcription PCR (qRT-PCR) has been engineered for sensitive amplification and detection of target RNAs via dynamically fluorescence monitoring [45][46][47][48]. qRT-PCR allows the reverse transcription of target RNA to its complementary DNA, which realizes the effective amplification and quantitative analysis of the target RNA [49]. In addition, to further develop more sensitive and accurate PCR technology, digital PCR (dPCR) was emerged to actualize the absolute quantification for nucleic acid detection and nucleotide copy number variant analysis [50].…”

A platform for multiplexed colorimetric microRNA detection using shape-encoded hydrogel particles

“…While there exist solution-based colorimetric assays with higher sensitivity (by using nucleic acid amplification), they cannot perform multiplexing. [11][12][13][14]45 Moreover, it is possible to increase the sensitivity of this platform by combining it with upstream nucleic acid amplification, which is compatible with hydrogels, such as rolling circle amplification or hybridization chain reaction. 46,47 These amplification schemes result in more biotin labels per target bound, resulting in more enzyme reporter labelling within the hydrogel particle.…”

“…[6][7][8][9][10] Conversely, solution-based colorimetric detection systems can realize fast hybridization kinetics but are not capable of simultaneous multiplexing. [11][12][13][14][15][16] Hydrogels offer a superior alternative to solution and surface-based biomolecule detection systems due to their biocompatibility, solution-like kinetics, non-fouling nature, and ability to incorporate capture agents inside a 3D network. 17,18 Hydrogels have previously been used to detect microRNA, mRNA, proteins and cytokines from a variety of complex samples such as total RNA extract, live and lysed cells, serum, and FFPE tissue.…”