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Reagent and method for detecting microRNA based on click chemical connection and hairpin stacking assembly

A technology of click chemistry and reagents, which is applied in biochemical equipment and methods, microbiological determination/inspection, etc. It can solve the problems of complex detection steps and achieve the effects of easy control, excellent performance and high sensitivity

Active Publication Date: 2021-10-29
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, how to convert a large number of ligation products into signal output needs to be considered. Most of the methods reported so far require the use of magnetic beads to separate or even re-use DNA polymerase, which makes the detection steps complicated again.

Method used

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  • Reagent and method for detecting microRNA based on click chemical connection and hairpin stacking assembly
  • Reagent and method for detecting microRNA based on click chemical connection and hairpin stacking assembly
  • Reagent and method for detecting microRNA based on click chemical connection and hairpin stacking assembly

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1, synthetic probe sequence

[0039] Referring to Table 1, the probes and other nucleic acid sequences used in the present invention were synthesized.

[0040] All nucleic acid sequences used in the present invention in table 1

[0041]

[0042]

[0043] Note: The underline in the table indicates the stem sequence of the DNA hairpin probe, and the italic indicates the loop sequence. The gray font indicates the mutated base site of let-7a.

Embodiment 2

[0044] Example 2. Combining click chemical ligation and hairpin stack assembly to detect microRNA

[0045] figure 1 The implementation process of the present invention is described in detail. When the target exists, two nucleic acid probes (N3 Probe, DBCOProbe) that are respectively complementary to the half sequence of the miRNA molecule Let-7a and are respectively modified with Azide and DBCO groups at the ends can Hybridize with the target so that the click chemical group is close to an efficient click chemical ligation reaction. When the temperature is raised to 95 degrees Celsius, the hybridization complex will be denatured and let-7a will be released, so that it can serve as the next reaction after the temperature is lowered to 30 degrees Celsius. template. After multiple cycles of heating and cooling, a small amount of targets can produce a large number of ligated products. Then we designed three exquisite metastable DNA hairpin structures HA, HB, and HC for the ligate...

Embodiment 3

[0048] Embodiment 3, sensitivity verification

[0049] The sensitivity of the detection method was verified, and the experimental results were as follows: image 3 As shown, in this detection strategy, when the target concentration is in the range of 20pM to 2nM, the fluorescence signal response shows a good linear relationship, and the linear fitting equation is △F=0.3336C+13.544, R 2 =0.9981, the lower detection limit reached 8.63pM. Further, in order to verify that the double-cycle signal amplification we proposed indeed has the effect of double signal amplification, we verified the sensitivity of a single hairpin stack assembly to detect microRNA, using "miR-analog (SEQ ID NO.14)" As a trigger target, results such as Figure 4 As shown, the linear range is 2nM-100nM, and the lower limit of detection is only 1.4nM, which further proves that in the present invention, the addition of the cyclic click chemistry ligation reaction amplifies the signal by more than 100 times an...

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Abstract

The invention discloses a reagent and a method for detecting microRNA based on click chemical connection and hairpin stacking assembly. The reagent for detecting microRNA comprises a target recognition probe and a hairpin probe, the target microRNA can be accurately separated from a homologous sequence, and the method does not need various enzymes to participate in the detection process; and the sensitivity is high, the target detection lower limit is 8.63 pM, the specificity is good, the serum adding standard recovery rate is 92.45%-108.92%, and a brand new thought is provided for applying the click chemical reaction and the enzyme-free DNA loop to the field of biological detection in the future.

Description

technical field [0001] The present invention relates to reagents and methods for detecting and quantifying nucleic acids, in particular to reagents and methods for detecting microRNA based on click chemical ligation and hairpin stacking assembly. Background technique [0002] MicroRNA is a kind of non-coding RNA with a length of 18 to 24 bases. It is secreted by living cells and plays an important role in the post-transcriptional regulation of genes. Therefore, it is related to many pathophysiological processes, such as cell differentiation and Apoptosis is closely related to organogenesis. A series of studies have found that microRNAs are abnormally expressed in the body fluids of patients suffering from tumors, cardiovascular diseases and even psychological disorders. Therefore, it is considered as a promising biomarker that can assist in disease diagnosis and treatment. [0003] However, due to the short sequence and low abundance of microRNA, the current conventional d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682
CPCC12Q1/682C12Q2525/207C12Q2525/301C12Q2533/107C12Q2537/1373C12Q2563/107
Inventor 赵爽陈鸣唱凯阳莎汤晓琦余恋雨
Owner ARMY MEDICAL UNIV
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