MiRNA-21 detection method

A miRNA-21 and detection method technology, which is applied in the field of microRNA detection, can solve the problems of complex operation, long detection time, and high requirements for operation skills, and achieves the effects of strong modifiability, low cost, and improved detection sensitivity.

Pending Publication Date: 2019-10-29
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The miRNA detection method in the prior art takes a long time to detect, and the operation is complicated. It also needs to use PCR to complete the detection, or the cooperation of fluorescence spectrophotometer, etc., requires relatively high operating skills of technicians

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Successful verification of substrate chain-sucrase complex and probe synthesis

[0039] 1. Preparation of substrate chain and sucrase linker complex:

[0040] Prepare 30 μL of substrate chain with a concentration of 1 mM in DEPC water, add 2 μL of sodium phosphate buffer (1M) at pH 5.5 and 2 μL of tris(2-carboxyethyl)phosphine (TCEP) at a concentration of 30 mM dissolved in Millipore water, and The mixture was allowed to stand at room temperature for 1 hour and then purified by ultrafiltration 8 times using Amicon-10K ultrafiltration tubes.

[0041] Sucrase-linked substrate chains: Prepare 400 μL of 20 mg / mL sucrase solution with buffer A, mix with 1 mg sulfo-SMCC; vortex for 5 minutes, place on a shaker at room temperature for 1 hour, and centrifuge at 1000r for 5 minutes Excess sulfo-SMCC was removed and the solution was purified by ultrafiltration 8 times with Amicon-100K and buffer A.

[0042] The purified sulfo-SMCC-activated sucrase solution was mixed...

Embodiment 2

[0051] Example 2: Detection sensitivity experiment of miRNA-21

[0052] 1. The instruments and reagents required for the experiment are the same as those in Implementation 1;

[0053] 2. Investigate the sensitivity of the deoxyribozyme and sucrase dual-enzyme amplification system to measure the miRNA-21 standard solution, including the following steps:

[0054] (1) Preparation of samples: prepare miRNA-21 standard solutions of 10pM-200pM concentration with DEPC water, so that the concentrations are 10pM, 20pM, 50pM, 100pM, 150pM, 200pM;

[0055] (2) Perform the reaction: put 20uL detection probe in the magnetic rack for 2 minutes, discard the clear solution, then add 10μL different concentrations of miRNA-21 to be detected (SEQ ID NO.2), 7μL DEPC-treated water, 2μL double Strand-specific nuclease buffer (containing 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2 and 1 mM DTT) and 1 μL DSN (1 U / uL). And react at 45°C for 50 minutes. The miRNA-21 to be tested specifically binds to the hyb...

Embodiment 3

[0061] Embodiment 3: Investigate the selectivity to miRNA-21

[0062] 1. The instruments, reagents and probe synthesis methods required for the experiment are consistent with those in Implementation 1;

[0063] 2. The sequences required for this experiment, in addition to the sequences applying for rights protection, also need to use the following sequences (as shown in Table 1):

[0064] Table 1 List of oligonucleotide sequences

[0065]

[0066]

[0067] 3. Deoxyribozyme and sucrase-based dual-enzyme amplification system measures the selectivity of miRNA-21 and mismatched sequences, including the following steps:

[0068] (1) Prepare samples: Prepare hsa-miR-200c, hsa-mir-423, hsa-miR-4640, has-miR-21, Mis-1, Mis-2, Mis-3 and NC sequences with DEPC water 200pM standard solution, blank control (Blank) is DEPC water;

[0069] (2) Perform the reaction: put 20 uL detection probe in the magnetic rack for 2 minutes, discard the clear solution, and then add 10 μL of differ...

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Abstract

The invention discloses a miRNA-21 detection method. According to the method, a substrate chain-sucrase complex is coupled on each magnetic bead, when a target miRNA appears, the target miRNA hybridizes with the substrate chains, double-strand specific nuclease can selectively cleave the DNA in DNA/RNA double strands and keep the activity of RNA, and the released miRNA is subjected to the next round of hybridizing cycle; and finally, the sucrase in supernatant hydrolyzes sucrose into glucose, and an output signal is obtained through the detection using a glucometer. The invention improves detection sensitivity by introducing the double-strand specific nuclease and the sucrase to carry out double-enzyme amplification on the detection signals of the miRNA, a linear relation is realized at 10-200 pM, a limit of detection is 1.8 pM, and the invention has the advantages that the detection method is simple and convenient, fast, sensitive, highly-efficient, etc.

Description

technical field [0001] The invention relates to a method for detecting microRNA, in particular to a method for detecting miRNA-21. Background technique [0002] microRNAs (miRNAs) are a class of endogenous non-coding small molecules with a length of about 18-25 nt, which exist in body fluids such as serum, plasma or urine. ) Complementary binding to regulate the expression of the subsequent gene at the translational level. Therefore, miRNAs participate in various physiological processes of organisms, controlling cell differentiation, proliferation and apoptosis. However, due to its specificity of low concentration in body fluids, short length and high homology, it is still challenging to achieve a fast, simple, reliable and ultrasensitive assay. [0003] Most of the existing miRNA detection methods require expensive instruments and equipment, complex operations, and long waiting times. Currently, there are four main methods for nucleic acid detection: southern and norther...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682C12Q1/54C12Q1/44C12Q1/34
CPCC12Q1/682C12Q1/54C12Q1/44C12Q1/34C12Q2525/207C12Q2521/301C12Q2563/143C12Q2563/149
Inventor 余伯阳田蒋为黄浠桐
Owner CHINA PHARM UNIV
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