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Kit for miRNA detection and application thereof

A technology for kits and detection primers, which is applied in the fields of medical detection and molecular biology, and can solve problems such as failure to meet detection requirements

Inactive Publication Date: 2019-03-22
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, in the study of chronic kidney disease, studies have shown that the expression level of miRNA-181a gene in the urine of patients is 200 times lower than that of healthy people, and the same result has been verified in the serum of patients with chronic kidney disease. The sensitivity of detection reagents puts forward high requirements, and traditional detection kits and methods can no longer meet the detection requirements of such extremely low concentrations of miRNA in samples

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  • Kit for miRNA detection and application thereof
  • Kit for miRNA detection and application thereof
  • Kit for miRNA detection and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Preparation of miRNA-181a detection kit

[0084] This embodiment provides a miRNA detection kit, which is composed as follows (as shown in Table 1): isothermal amplification reaction buffer, primer mixture, enzyme system, mineral oil, physiological saline, digital microfluidic chip and instructions.

[0085] Preparation of digital microfluidic chip: The preparation material of digital microfluidic chip is mainly polydimethylsiloxane, and the preparation raw material of digital microfluidic chip is prepared according to the following mass ratio: polydimethylsiloxane: curing agent Sylguard-B:Triton X-100=10:1:0.002.

[0086] After the above-mentioned preparation raw materials are mixed, pour into mold (such as figure 1shown), baked at 80°C for 2 hours, demolded, punched, and then cleaned by plasma, so that the digital microfluidic substrate and the glass sheet were fully bonded, and the digital microfluidic chip was prepared; the digital microfluidic chip Phys...

Embodiment 2

[0099] Example 2 Sensitivity analysis of miRNA-181a detection kit

[0100] This example analyzes the sensitivity of the miRNA-181a detection kit prepared in Example 1, specifically: using human miRNA-181a as the target sequence (as shown in SEQ ID NO.5), different concentrations of human miRNA-181a Absolute quantitative analysis was carried out on the standard, the specific method is as follows:

[0101] 1. The lyophilized powder of the synthesized human miRNA-181a template was serially diluted to obtain three concentrations of miRNA-181a standards, which were 100fM, 10fM, and 1fM respectively, and a negative control group using water as a template was set at the same time.

[0102] 2. Prepare reagents in the reagent preparation room according to the instructions of the kit. Isothermal amplification reaction buffer 1 is 16 μL×4, primer mixture 2 is 2 μL×4, enzyme system 3 is 1 μL×4, and the reaction solution is 19 μL / The tubes are divided into EP reaction tubes, and the reac...

Embodiment 3

[0114] Example 3 Specific Analysis of miRNA-181a Detection Kit

[0115] In this example, the miRNA-181a mutant sequence (as shown in SEQ ID NO.6) containing one base difference is used as the target sequence, and absolute quantitative analysis is performed on the standard substance of the mutant sequence at a specific concentration, and the same concentration of miRNA Use the -181a standard as a reference to observe whether there is an amplification signal in the mutant sequence detection group. The specific method is as follows:

[0116] 1. Dilute the standard product of the miRNA-181a mutant sequence to 10fM, and at the same time dilute the miRNA-181a standard product to the same concentration (10fM).

[0117] 2. Prepare reagents in the reagent preparation room according to the instructions of the kit. Isothermal reaction buffer 1 is 16 μL×2, primer mixture 2 is 2 μL×2, enzyme system 3 is 1 μL×2, and the reaction solution is divided into 19 μL / tube. Put it into the EP react...

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Abstract

The invention provides a kit for miRNA detection and application thereof. The kit comprises a miRNA detection primer, DNA polymerase, incising endonuclease and a digital microfluidic chip. The kit isdeveloped based on the digital microfluidic chip and an isothermal amplification technology, and has relatively high detection sensitivity, specificity and accuracy. At the same time, the kit has relatively strong universality, and the detection of all miRNA can be realized only by designing a specific base sequence of a primer according to specific sequences of different miRNA. The kit provided by the invention directly can be used for directly detecting free and trace miRNA in body fluid without complicated pretreatment, RNA extraction, reverse transcription and the like on a human body fluid sample, is not interfered by other similar miRNA sequences, can realize absolute quantification of miRNA, has the advantages of simple operation, low requirement on detection equipment, low cost andthe like, and is suitable for wide popularization and application.

Description

technical field [0001] The invention relates to the technical fields of medical detection and molecular biology, in particular to a kit for detecting miRNA and its application. Background technique [0002] MicroRNA (miRNA) is a type of endogenous non-coding single-stranded RNA found in a variety of eukaryotic cells and viruses, with a length of about 18-24 nucleotides and a high degree of conservation in evolution . miRNAs do not encode proteins, but can regulate gene expression post-transcriptionally and affect protein translation. Studies have shown that one miRNA can regulate the expression of one or more genes, and multiple miRNAs can also jointly regulate the same gene, interrupting its translation and preventing the synthesis of corresponding proteins, thereby realizing the regulation of cell functions. Based on this regulatory function, miRNA plays an important role in important physiological and pathological processes such as cell development, apoptosis, different...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2531/119C12Q2563/107C12Q2565/629C12Q2521/301C12Q2537/1376
Inventor 赵俊朱灵刘勇邓国庆朱灿灿崔俊生杨柯周喃
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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