Method for detecting miRNA-155 based on aptamer

A nucleic acid aptamer, mir-155 technology, applied in the field of biotechnology detection, can solve the problems of difficulty in satisfying miR-155 convenient, fast, high-sensitivity detection, complex sample preprocessing, low detection sensitivity, etc., and achieves fast detection speed. , low detection limit, simple preparation method

Active Publication Date: 2017-06-23
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently reported detection methods for miR-155 include real-time quantitative PCR, Northern blot hybridization, and gene chip detection. It is difficult to meet the requirements of convenient, fast and highly sensitive detection of miR-155

Method used

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  • Method for detecting miRNA-155 based on aptamer
  • Method for detecting miRNA-155 based on aptamer
  • Method for detecting miRNA-155 based on aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042]a. Mix 1 μL of different concentrations of Hairpin1 (final concentrations are 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM, 1.2 μM, 1.4 μM), Circular template1 (1 μL, 10 μM), Hairpin2 (1 μL, 10 μM), Circular template 2 (1μL, 10μM), Molecular beacon (1μL, 100μM), phi29 DNA polymerase (1μL, 10×), Nb.BbvCI (1μL, 10×), NEB buffer (5μL, 10×) and the test substance (miRNA-155) were added to the EP tube, shaken for 30s, and placed in a water bath at 37°C for 70min.

[0043] b. Take the reacted solution out of the water bath, put it into a water bath at 85°C and heat it in a water bath for 10 minutes to inactivate the enzyme in the system.

[0044] c. After 10 min, take out the mixed solution from the water bath. Then an Agilent fluorescence detector was used to measure the fluorescence intensity of the mixed solution, and the target object was detected accordingly.

[0045] Its experimental principle diagram is as figure 1 , see the result figure 2 , it can be seen from the figure tha...

Embodiment 2

[0047] a. Mix 1 μL of different concentrations of Circular template1 (final concentrations are 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM, 1.2 μM, 1.4 μM), Hairpin1 (1 μL, 10 μM), Hairpin 2 (1 μL, 10 μM), Circular template 2 (1μL, 10μM), Molecular beacon (1μL, 100μM), phi29 DNA polymerase (1μL, 10×), Nb.BbvCI (1μL, 10×), NEB buffer (5μL, 10×) and the test substance (miRNA-155) were added to the EP tube, shaken for 30s, and placed in a water bath at 37°C for 70min.

[0048] b. Take the reacted solution out of the water bath, put it into a water bath at 85°C and heat it in a water bath for 10 minutes to inactivate the enzyme in the system.

[0049] c. After 10 min, take out the mixed solution from the water bath. Then an Agilent fluorescence detector was used to measure the fluorescence intensity of the mixed solution, and the target object was detected accordingly.

[0050] see results image 3 , it can be seen from the figure that the detected fluorescence intensity value increases as...

Embodiment 3

[0052] a. Add 1 μL of different concentrations of Hairpin2 (0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM, 1.2 μM, 1.4 μM), Hairpin1 (1 μL, 10 μM), Circular template 1 (1 μL, 10 μM), Circular template 2 (1μL, 10μM), Molecular beacon (1μL, 100μM), phi29 DNA polymerase (1μL, 10×), Nb.BbvCI (1μL, 10×), NEB buffer (5μL, 10×) and the test substance (miRNA-155) were added to the EP tube, shaken for 30s, and placed in a water bath at 37°C for 70min.

[0053] b. Take the reacted solution out of the water bath, put it into a water bath at 85°C and heat it in a water bath for 10 minutes to inactivate the enzyme in the system.

[0054] c. After 10 min, take out the mixed solution from the water bath. Then an Agilent fluorescence detector was used to measure the fluorescence intensity of the mixed solution, and the target object was detected accordingly.

[0055] see results Figure 4 , it can be seen from the figure that the detected fluorescence intensity value increases as the concentration of Ha...

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Abstract

The invention provides a method for detecting miRNA-155 based on an aptamer. The biological detection method is established based on specific recognition of the aptamer and a target object by means of the chain extension effect of phi29 DNA polymerase in rolling circle amplification, the cutting action of endonuclease on a nucleic acid chain at a specific site, and the fluorescence signal generating characteristic of a fluorophore of a molecular beacon. The detection method has the advantages that the detection speed is high, the detection limit is low, and specificity is high. Defects of existing miRNA-155 detection methods can be overcome, and quick and accurate quantitative detection of miRNA-155 can be achieved.

Description

technical field [0001] The invention relates to the technical field of biotechnology detection, in particular to a method for detecting miR-155 based on nucleic acid aptamers. Background technique [0002] MiRNA is a class of non-coding RNA molecules with a length of 17-25 nucleotides, which play an important role in life phenomena such as tumorigenesis, cell differentiation, apoptosis and protein synthesis. The abnormal expression of miRNA is directly related to various human diseases such as cancer, diabetes, cardiovascular disease and neurological diseases, so miRNA has become an important biomarker for early prevention and diagnosis of various diseases such as cancer. miRNA-155 is a kind of miRNA, and its overexpression will lead to the occurrence of breast cancer. Therefore, it is very necessary to realize the early diagnosis and prevention of breast cancer by highly sensitive detection of miR-155. [0003] Currently reported detection methods for miR-155 include rea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2521/101C12Q2521/301C12Q2563/107C12Q2531/125
Inventor 李慧徐成功王玉
Owner UNIV OF JINAN
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