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Digital miRNA detection method, detection reagent and detection kit

A technology of reagents and molecular beacons, applied in the field of detection reagents and detection kits, and digital miRNA detection methods, can solve the problems of difficult application of digital detection, low efficiency, and high detection cost

Active Publication Date: 2020-07-07
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is worth noting that miRNAs are usually short and highly homologous, with only minor differences between them. These characteristics make commonly used PCR amplification techniques inaccurate and inefficient. At the same time, PCR replication of miRNAs requires tailing primers, Difficult to apply to digital detection and the specificity is not high, and the cost of detection is relatively high; Northern blotting is very complicated, requires radioactive labeling, and will cause serious pollution, low detection efficiency, and the detection limit is not ideal, so it is difficult to apply to actual samples Trace miRNA detection; microarrays have good detection limits, but the required equipment and material costs are high, thus limiting the use of this method; many new biosensors have also been developed for miRNA detection, the principle includes surface-enhanced pull Mann scattering, electrochemistry, surface plasmon resonance, colorimetry, electrochemiluminescence, quartz crystal microbalance, etc. However, these methods based on nucleic acid hybridization have insufficient detection limit or detection cost methods. Due to these limitations, it is more effective to develop and low-cost detection methods are meaningful and important
[0005] miRNA is an important target in the field of chemical biology research. Traditional detection methods cannot meet the current needs, so new methods with higher sensitivity and specificity, simpler and lower cost need to be developed

Method used

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  • Digital miRNA detection method, detection reagent and detection kit
  • Digital miRNA detection method, detection reagent and detection kit
  • Digital miRNA detection method, detection reagent and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The hairpin-like molecular beacon probe of the present invention needs to be heated to 95 degrees Celsius after adding ultrapure water to completely melt into a linear single chain, and then slowly annealed to 55 degrees or room temperature to ensure the formation of a hairpin-like structure.

[0055] The out-of-droplet detection is performed first. Take 50pmol molecular beacon (total 10μl), 50pmol primer (10μl), 80μl reaction solution, 10mM Tris-HCl, 50mM NaCl, 10mM MgCl 2 , 1mM DTT, 10units Klenow fragment, 5nmol dNTP, mixed the above solutions (total 100μl), mixed with 10μl of different miRNA solutions, reacted at 37 degrees for 2-4h, detected the emission fluorescence of FAM, carried out three groups of experiments, the results As follows. All nucleic acid molecules involved in the experiment were dissolved in ultrapure water and prepared as a solution with a concentration of 10 mM.

[0056] Table 1 gives all nucleic acid sequences. When the control RNA molecule ...

Embodiment 3

[0067] Take 20mM Tris buffer (pH 7.4, containing 100mM NaCl, 5mM KCl and 5mM MgCl 2 ), adding different concentrations of target miRNA (as shown in SEQ ID No.2), the final concentration of miRNA is between 2 and 20fM.

[0068] Microfluidic chips based on PDMS and glass substrates were processed by soft lithography. The chip contains 3 input channels, one of which is fed into the oil phase using a micro-injection pump, and the other 2 channels are fed into the water phase. The oil phase uses Novec7500, and adds surfactant pico-surf (2% w / w), which can make the generated droplets stable and not easy to crack or fuse with each other. One of the water phase channels was passed into the above miRNA solution, and a blank control group without miRNA was set at the same time; the other water phase channel was passed into Tris buffer, which contained 10mM Tris-HCl, 10mM MgCl 2 , the Klenow fragments (Klenow fragments) of 50mMNaCl, 1mM DTT, 5Units, the dNTPs of 1nmol, the hairpin type...

Embodiment 4

[0076] Experimental conditions and methods are the same as in Example 1.

[0077] table 5

[0078]

[0079] The detection results of comparative PCR are respectively the target miRNA, the comparison RNA with one mismatched base and the blank control. It can be seen that the specificity of this method is much higher than that of PCR.

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Abstract

The present invention relates to the field of nucleic acid detection and particularly to a digital miRNA detection method, a detection reagent and a detection kit. The provided detection method utilize entire sequence information of a target miRNA, combines with a droplet microfluidic technology, constructs a digital miRNA detection platform, and has relatively high specificity and ability to identify single base mismatches. The platform can quantify miRNA in a concentration range of 2-20 fM at a single molecule level and proves effectiveness and practicability of the method. For the first time, highly selective direct digital miRNA detection is realized.

Description

technical field [0001] The invention relates to the field of nucleic acid detection, in particular to a digital miRNA detection method, a detection reagent and a detection kit. Background technique [0002] MicroRNA (miRNA) detection is of great significance in disease diagnosis and miRNA function research. The importance of miRNA itself lies in its complex regulatory functions in various life processes and its close relationship with certain diseases. [0003] miRNAs are short single-stranded non-coding RNAs ranging in length from 18-25 nt that are present in eukaryotic cells. They are produced from hairpin-like precursors that base-pair to the corresponding target mRNA in the RNA-induced silencing complex (RISC). miRNAs cause degradation of mRNAs or inhibit their translation due to base pairing of complete or incomplete complementarity to their targets. miRNA expression levels vary in different tissues and organs and are precisely regulated; thus, dysfunction of their e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6886C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2600/178C12Q2563/159C12Q2537/1373C12Q2563/107C12Q2525/207C12Q2565/629
Inventor 任大海王斌尤政李静
Owner TSINGHUA UNIV