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MiRNA-208a amplification primer pair based on chain exchange amplification and detection kit thereof

1. The technology of mirna-208a and primer pair is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc. It can solve the problems of high cost of instruments and reagents, poor sensitivity, complex design, etc., and achieve the goal of instrument Simple, highly specific, non-polluting effects

Pending Publication Date: 2021-10-08
SOUTHEAST UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional Northern blotting is less sensitive, time-consuming and laborious, and requires a large amount of RNA samples and radioactive probes
The microarray hybridization method involves the modification of probes, the production of chips, and the labeling of miRNA, etc., which is costly and complicated to design.
The commonly used RT-qPCR method has high sensitivity and specificity, but the cost of instruments and reagents is high, and well-trained operators are required, so it is difficult to be widely used in primary medical units and on-site testing

Method used

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  • MiRNA-208a amplification primer pair based on chain exchange amplification and detection kit thereof
  • MiRNA-208a amplification primer pair based on chain exchange amplification and detection kit thereof
  • MiRNA-208a amplification primer pair based on chain exchange amplification and detection kit thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1: Preparation of fluorescence immunochromatography quantitative detection test strip

[0047] Fluorescence immunochromatography quantitative detection test strip, which consists of a base plate, absorbent paper, nitrocellulose membrane, binding pad, sample pad, the base plate is pasted with nitrocellulose membrane, one end of the nitrocellulose membrane is connected to the absorbent paper, and the other end is connected to the binding pad Lap the sample pad on the binding pad, and the nitrocellulose membrane is provided with a quality control line (C) that is coated with a kind of biotinylated bovine serum albumin, and is also provided with a quality control line parallel to the quality control line (C). The detection line (T) of anti-digoxigenin antibody; the binding pad is a glass fiber membrane immobilized with fluorescent microspheres labeled with streptavidin; the test strip also includes a cartridge, which is provided with a sample window and a signal r...

Embodiment 2

[0053] Example 2: Design and Screening of Amplification Primers Based on miRNA-208a

[0054] According to the published miRNA-208 reference sequence (NC000014.9) in the gene sequence database GenBank of the American Center for Biotechnology Information, use BLASTn, MAFFT, DNASTAR and other molecular biology tools for genome sequence comparison analysis to obtain the sequence specificity of miRNA-208a Conserved region (miRNA-208a-3p). Based on the conserved target sequence, Primer Premier 5.0 and the online tool NUPACK (http: / / www.nupack.org / ) were used to design isothermal amplification primers. The designed primers were further verified by BLASTn and Primer-BLAST searches; the sequences of the designed primers were as follows:

[0055] Table 1 Target gene and isothermal amplification primer information

[0056]

[0057] The isothermal amplification reaction system and procedure are as follows: use miRNA-208a standard substance (according to the sequence of miRNA-208a-3p,...

Embodiment 3

[0060] Example 3: Establishment of a fluorescent quantitative detection method for miRNA-208a sequence based on isothermal amplification

[0061] Use the miRNA-208a standard substance (miRNA-208a-3p) (10mM) synthesized at HPLC level (entrusted to Biotechnology (Beijing) Co., Ltd. to synthesize) and negative control (normal saline) for isothermal amplification method testing to optimize Reaction temperature and reaction time, concrete steps are as follows:

[0062] 1. Determination of reaction temperature

[0063] The standard substance (1×10 10 pmol / L) as a template, isothermal amplification was carried out under the action of miRNA-208a-specific primers obtained in Example 2, wherein the 20 μL reaction system included: upstream and downstream primers Primer-F (10mM) and Primer-R (10mM) 1.5 μL each, 2 μL of 10× isothermal amplification buffer, Bst 2.0 WarmStart TM DNA polymerase (8000U / mL) 0.8μL, liquid polyethylene glycol (molecular weight 200-600) 2μL, dNTPs (1-10mM) 2.5...

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Abstract

The invention discloses a miRNA-208a amplification primer pair based on chain exchange amplification and a detection kit thereof. The amplification primer pair comprises a forward primer and a reverse primer; the forward primer is composed of a 5' end side balance sequence and a 3' end side amplification sequence, and the amplification sequence is homologous with the 5' end side sequence of miRNA-208a-3p; and the reverse primer is composed of a 5' end side amplification sequence and a 3' end side balance sequence, and the amplification sequence is complementary with the 3' end side sequence of the miRNA-208a-3p. After an isothermal amplification reaction of the miRNA-208a is completed, a running buffer solution is added, the mixture is uniformly mixed and dropwise added to a sample adding window of a test strip, and a fluorescence signal is detected after 2 minutes. The detection method provided by the invention can be completed within 1 hour. The miRNA-208a detection method is simple, convenient, rapid and safe to operate, and an early molecular diagnosis detection method can be provided for acute myocardial infarction.

Description

technical field [0001] The invention relates to a miRNA-208a amplification primer based on strand exchange amplification and a detection kit thereof, and belongs to the technical fields of molecular biology detection technology, lateral flow chromatography technology and the like. Background technique [0002] The morbidity and mortality of cardiovascular diseases are increasing year by year, which greatly threatens the life and health of the people. The number of patients with cardiovascular disease in my country is about 330 million. Cardiovascular disease ranks first in the total death causes of urban and rural residents, and the prevention and treatment of cardiovascular disease is imminent. Coronary heart disease is a kind of cardiovascular disease, and acute myocardial infarction (Acutemyocardial infarction, AMI) is the main clinical manifestation of coronary heart disease attack. When cardiomyocytes are damaged, they will release proteins, exosomes, etc. into the per...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q2531/119C12Q2537/1376C12Q2563/107C12Q2525/207C12Q2565/625C12Q2545/113
Inventor 张宇田培龙庄林林王路海马明顾宁王建国
Owner SOUTHEAST UNIV
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