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A method for detecting microRNA based on helicase-dependent DNA constant temperature amplification technology

A constant temperature amplification and technical detection technology, applied in the field of biological analysis, can solve the problems of microRNA detection that have not yet been seen, and achieve the effects of high specificity, high detection sensitivity, and background signal reduction

Active Publication Date: 2020-03-27
SHANDONG NORMAL UNIV
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  • Claims
  • Application Information

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Problems solved by technology

At present, there are few reports on HDA detection methods. The relevant research mainly uses HDA technology to detect some pathogenic bacteria, but there is no report using HDA method to detect microRNA.

Method used

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  • A method for detecting microRNA based on helicase-dependent DNA constant temperature amplification technology
  • A method for detecting microRNA based on helicase-dependent DNA constant temperature amplification technology
  • A method for detecting microRNA based on helicase-dependent DNA constant temperature amplification technology

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Embodiment 1

[0058]Extraction of total RNA in cells: the culture medium of human cervical cancer cells (HeLa) was Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, They were cultured in an incubator at 37°C containing 5% carbon dioxide. When the cells grow to the logarithmic growth phase, the total RNA in the cells is extracted and purified using the total RNA mini-extraction kit (Catalog No. 74104) of the German Qiagen Biotechnology Company. The extraction and purification operations are strictly in accordance with the instructions attached to the kit. Instructions are carried out. The obtained total RNA was used for the quantitative detection of microRNA in cancer cells after the concentration was measured using a UV-Vis spectrophotometer.

[0059] Exonuclease I (Exo I) digestion: first prepare 19 μl of reaction buffer containing 1 mmol / L of dithiothreitol (DTT), 1× exonuclease I (Exo I) reaction buffer, 20 units of RNase ...

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Abstract

The invention discloses a helicase-dependent isothermal DNA amplification-based method for detecting microRNA. The method comprises the following steps: (1) extracting total RNA in a sample; (2) adding a single-strand DNA probe and excessive exonuclease I into the extracted total RNA, incubating in a reacting buffer solution to realize specific combination of target microRNA and the single-strand DNA probe, and removing residual single-strand DNA probe by utilizing the excessive exonuclease I; and (3) adding upstream primer, downstream primer, single-strand binding protein and helicase to the reaction system, amplifying the target microRNA, and detecting expression of the target microRNA through a fluorescent signal. According to the method, the background is reduced by digesting the exonuclease I, and the signal is amplified by means of exonuclease assisted isothermal amplification (HDA) reaction, so that the microRNA can be quickly and sensitively detected.

Description

technical field [0001] The invention relates to the technical field of biological analysis, in particular to a method for detecting microRNA based on a helicase-dependent DNA constant temperature amplification technology. Background technique [0002] microRNA is a type of endogenous non-coding single-stranded RNA discovered in a variety of eukaryotic cells and viruses in recent years. It is a short sequence of 21-25 nt in length and is highly conserved in evolution. Complementary base pairing can cause the degradation of target mRNA or inhibit its translation, thereby regulating the expression of genes after transcription. Because the microRNA sequence has a certain degree of conservation in different organisms, it is generally believed that the function of microRNA is to participate in some basic processes of life, such as cell proliferation, cell death, stress response and fat metabolism during development. [0003] Therefore, microRNA is considered as a potential biomar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2521/513C12Q2522/101C12Q2521/319
Inventor 张春阳马飞刘萌
Owner SHANDONG NORMAL UNIV
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