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MicroRNA trace detection method based on exponential order non-enzymatic amplification and electrochemical luminescence principle

A non-enzymatic amplification and detection method technology, applied in the field of microRNA trace detection, can solve the problem of low sensitivity and achieve the effect of specificity guarantee

Inactive Publication Date: 2015-03-25
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, Chinese patent 201310294770.7 discloses a microRNA detection method based on the principle of non-enzyme amplification and electrochemiluminescence. This method realizes a constant temperature, non-enzyme amplification microRNA detection method, but the sensitivity of this method is low

Method used

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  • MicroRNA trace detection method based on exponential order non-enzymatic amplification and electrochemical luminescence principle
  • MicroRNA trace detection method based on exponential order non-enzymatic amplification and electrochemical luminescence principle
  • MicroRNA trace detection method based on exponential order non-enzymatic amplification and electrochemical luminescence principle

Examples

Experimental program
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Effect test

Embodiment 1

[0069] Example 1 Construction of exponential signal enhanced non-enzyme amplification platform and its feasibility verification

[0070] According to the principle of the above-mentioned exponential signal enhanced non-enzymatic amplification platform, the hairpin probes H1, H2, H3, and H4 required for the microRNA21 sequence were designed for the non-enzymatic amplification system. Their sequences are shown in Table 1, and their two-dimensional structures are shown in Table 1. figure 2 shown. The 5' and 3' ends of hairpin probes H1, H3, and H4 are labeled with six carbon amino groups, and the corresponding 3' ends of H2 are labeled with biotin. The hairpin probes were all synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd. H1, H3, and H4 need to be labeled with electrochemiluminescent groups, while H2 can be dissolved in TE buffer (final concentration 10 μM, stored in -20 refrigerator) . The labeling process of H1, H3, and H4 is as follows: take 2.5OD hairpin probe, ...

Embodiment 2

[0076] Example 2 The establishment of exponential signal enhanced non-enzyme amplification platform and its sensitivity experiment

[0077]The establishment of the exponential signal enhanced non-enzymatic amplification platform includes the establishment of the amplification system and the detection of electrochemical signals. The establishment of the amplification system is shown in Example 1. The main steps of electrochemiluminescence signal detection are as follows: add streptavidin-coated magnetic beads (final concentration 0.2 mg / mL) to the non-enzymatic amplification system, incubate at 37°C for 30 minutes, separate with a magnetic separator, and remove the solution Afterwards, 100 μL of PBS buffer solution (final concentration: 1×) was added to dissolve, and then separated, thereby repeatedly washing three times. Finally, the product was put into a Roche Elecsys 2010 electrochemiluminescence automatic immunoassay analyzer to detect the electrochemiluminescence signal. ...

Embodiment 3

[0079] Example 3 Exponential signal enhanced non-enzyme amplification platform specificity verification

[0080] In order to verify the specificity of the exponential signal enhancement non-enzymatic amplification platform, microRNA210 and microRNA214 were added to the platform (the amount added was 10 pmol respectively), and the electrochemiluminescent signal was detected. The experimental results are as follows Figure 5 shown. Depend on Figure 5 It can be seen that the control group is a blank control, the electrochemiluminescence signals of the microRNA210 and microRNA214 experimental groups are the same as those of the control group, and the experimental group obtains stronger electrochemiluminescent signals. Therefore, the platform specificity is better.

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Abstract

The invention discloses a microRNA trace detection method based on an exponential order non-enzymatic amplification and electrochemical luminescence principle. A non-enzymatic amplification and hybrid chain type reaction system is adopted, and the specific sequences of DNA hairpin probes H1, H2, H3 and H4 are designed based on a detection target microRNA sequence; when an amplification system contains to-be-detected microRNA, the subsequent hybrid chain type reaction process is triggered by virtue of an H1+H2 double-chain composite structure, and is finished by H3 and H4 together; and moreover, an amplification product is captured by virtue of streptavidin magnetic bead capture, and an electrochemical luminescence signal is generated and detected by virtue of an electrochemical detection system. According to the method, an enzyme is not involved in the whole process, the principle is simple, and the detection cost is low; and the method has the advantages of constant temperature amplification, high sensitivity, simple operation, simplicity in popularization and the like. The method is applied to nucleic acid detection and can be combined with a protein aptamer related technology to be used for protein detection.

Description

technical field [0001] The invention belongs to the technical field of electrochemical trace detection of nucleic acid, in particular to a microRNA trace detection method based on an exponential signal enhanced non-enzyme amplification platform combined with the principle of electrochemiluminescence. Background technique [0002] microRNA (miRNA) is a class of non-coding, endogenous microRNA with a length of 19-25 nucleotides, which is widely distributed in eukaryotic cells and participates in important life processes. In recent years, with the advancement of molecular biology and related disciplines, researchers have discovered hundreds of miRNAs and determined the main functions of some of them. This type of RNA mainly performs the function of gene regulation. After the non-coding sequence is transcribed into primary transcripts, the primary transcripts are produced by cleavage and processing of different nucleases. According to the researchers' speculation, about 30% of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/76
CPCC12Q1/6823G01N21/763C12Q2563/179C12Q2563/143C12Q2525/207
Inventor 周小明邢达廖玉辉
Owner SOUTH CHINA NORMAL UNIVERSITY
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