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Method for detecting miRNA-21 by blood glucose meter based on dnazyme and sucrase

A technology of miRNA-21 and deoxyribozyme, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of miRNA high NGS error rate, limited clinical application, lengthy time, etc., to improve detection sensitivity, Highly modifiable, low-cost effects

Pending Publication Date: 2019-09-17
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the relatively low selectivity during reverse transcription and PCR, it is more suitable for quantification of longer DNA / RNA targets, less sensitive for 20 base miRNAs, and requires finer control of temperature
Novel NGS systems offer greater detection dynamic range than PCR, but miRNA sequencing is still limited by relatively high NGS error rates
In addition, NGS has high requirements on infrastructure, computer capacity and personnel expertise, which limits its clinical application, and the cost of one test is very high
[0004] The Chinese invention patent with the notification number CN107385014A discloses a qRT-PCR method for direct quantitative detection of miRNA. A Chinese invention patent discloses a miRNA detection method based on the fluorescence amplification of rare earth nanomaterials, which also requires the use of a fluorescence spectrophotometer for quantification, and has certain professional requirements for the operating skills of the experimenters

Method used

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  • Method for detecting miRNA-21 by blood glucose meter based on dnazyme and sucrase
  • Method for detecting miRNA-21 by blood glucose meter based on dnazyme and sucrase
  • Method for detecting miRNA-21 by blood glucose meter based on dnazyme and sucrase

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Embodiment 1

[0039] Example 1: Feasibility verification of the detection method of the present invention, evaluation of the competition and hybridization feasibility of the system design sequence in a buffer solution

[0040] 1. Preparation of substrate strand and sucrase-linked complex

[0041] 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.

[0042] 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 ...

Embodiment 2

[0053] Embodiment 2: investigate the sensitivity of the present invention to miRNA-21

[0054] 1. The instruments and reagents required for the experiment are the same as those in Implementation 1;

[0055] 2. Investigate the sensitivity of the deoxyribozyme and sucrase dual-enzyme amplification system to measure the miRNA-21 standard solution, including the following steps:

[0056] (1) Preparation of samples: prepare standard solutions of miRNA-21 at concentrations of 100fM-1000fM with DEPC water, so that the concentrations are 100fM, 200fM, 400fM, 600fM, 800fM, and 1000fM;

[0057] (2) Start the reaction: Add 10 μL miRNA-21 standard solution and 60 μL probe dissolved in buffer C to each tube, and then add Mn dissolved in DEPC water at a concentration of 0.01M 2+ solution; incubate at room temperature for 40min;

[0058] (3) Stop the reaction: Add 5 μL of 50 mM EDTA to stop the reaction;

[0059] (4) Magnetic separation: Adsorb the magnetic beads with a magnet, transfer t...

Embodiment 3

[0064] Embodiment 3: investigate the selectivity of the present invention to miRNA-21

[0065] 1. The instruments, reagents and probe synthesis methods required for the experiment are consistent with those in Implementation 1;

[0066] 2. The sequences required for this experiment, in addition to the sequences applying for rights protection, also need to use the following sequences:

[0067] name oligonucleotide sequence has-mir-200c 5'-cgtcttacccagcagtgtttgg-3' hsa-mir-423 5'-tgaggggcagagagcgagacttt-3' hsa-miR-4640 5'-tgggccagggagcagctggtggg-3' has-miR-21 5'-uagcuuaucagacugauguuga-3' Mis-1 5'-uaccuuaucaacacugaucuuga-3' Mis-2 5'-uagcuuaucagacagauguuga-3' Mis-3 5'-uagcuuaacagacugauguuga-3'

[0068] 3. Deoxyribozyme and sucrase-based dual-enzyme amplification system measures the selectivity of miRNA-21 and mismatched sequences, comprising the following steps:

[0069] (1) Preparation of samples: Prepare hsa-miR-20...

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Abstract

The invention discloses a method for detecting miRNA-21 by a blood glucose meter based on dnazyme and sucrase. The method comprises the following steps: coupling a substrate chain and a dnazyme chain silenced by a sway chain on a magnetic bead, allowing target miRNA to be hybridized and combined with the sway chain after the target miRNA appears, and separating from the dnazyme chain so as to activate the dnazyme to cut the substrate chain; and allowing the released sucrase to hydrolyze sucrose to glucose, and acquiring the output signal by the detection of a blood glucose meter. According to the method, the detection signal of the miRNA is subjected to double enzyme amplification by introducing the dnazyme and the sucrase, so that the detection sensitivity is improved, a linear relation exists in the range of 100-1000fM, the detection limit is 68.08fM, and the method has the advantages of simplicity, convenience, rapidness, sensitivity, high efficiency, low cost and the like.

Description

technical field [0001] The invention relates to a method for detecting miRNA-21, in particular to a method for detecting miRNA-21 with a blood glucose meter based on deoxyribozyme and sucrase. Background technique [0002] MicroRNA (miRNA) widely exists in eukaryotic cells and is a short-length (about 18-25 nucleotides) endogenous non-coding small molecule, which is involved in the regulation of gene expression at the post-transcriptional level after binding to messenger RNA . miRNA regulates about 1 / 3 of the gene expression in the human body and participates in the control of 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682
CPCC12Q1/682C12Q2525/207C12Q2521/345C12Q2563/131C12Q2563/143C12Q2563/149
Inventor 余伯阳田蒋为黄浠桐
Owner CHINA PHARM UNIV
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