Construction method of magnetic microsphere DNA probe for detecting miRNA molecules as well as product and application of magnetic microsphere DNA probe

A technology for DNA probes and construction methods, applied in the direction of recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problem of inability to directly detect miRNAs levels and non-specific amplification, diagnostic methods cannot reflect miRNAs levels, In order to solve the problems of large sample size and other problems, the separation speed and enrichment efficiency can be improved, non-specific amplification can be avoided, and the method can be simplified.

Pending Publication Date: 2020-08-18
CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They have their own advantages and disadvantages: the method without amplification is easy to operate, but requires a large sample size and low sensitivity; while the method based on amplification has high sensitivity, but it is limited by the inability to directly detect the level of miRNAs and non-specific heterosexual expansion
[0005] Therefore, the existing diagnostic methods cannot reflect the real level of miRNAs, and the expression of miRNAs in body fluids is much lower than that in tissues, so the techniques for conventional detection of miRNAs in tissues are not necessarily applicable to the detection of miRNAs in body fluids. For detection, it is necessary to pay attention to new probes and methods that can enhance the detection signal

Method used

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  • Construction method of magnetic microsphere DNA probe for detecting miRNA molecules as well as product and application of magnetic microsphere DNA probe
  • Construction method of magnetic microsphere DNA probe for detecting miRNA molecules as well as product and application of magnetic microsphere DNA probe
  • Construction method of magnetic microsphere DNA probe for detecting miRNA molecules as well as product and application of magnetic microsphere DNA probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Preparation of DNA probes:

[0033] According to the molecular sequence (SEQ ID NO: 1) of the lung cancer marker miRNA-21, four kinds of DNA probes P1, P2, P3 and P4 that are paired with the sequence but have different modified molecules were synthesized, and the specific structures are shown in Table 1 .

[0034] Table 1 DNA probes modified with different groups

[0035] probe molecule Sequence (from 5' to 3' end) modify P1 (SEQ ID NO: 2) TC AAC ATC AGT CTG ATA AGC TA 5'cy3,3'biotin TEG P2 (SEQ ID NO: 3) TC AAC ATC AGT CTG ATA AGC TA 5'cy3,3'biotin (T) P3 (SEQ ID NO: 4) TC AAC ATC AGT CTG ATA AGC TA 5'cy3,3'biotin P4 (SEQ ID NO: 5) TC AAC ATC AGT CTG ATA AGC TA 5'biotin TEG,3'cy3

[0036] 2. Preparation of magnetic microsphere DNA probe:

[0037] (1) Superparamagnetic microspheres and streptavidin are covalently combined to form streptavidin magnetic beads (streptavidin MBs, SMBs). 8.0, 0.5M NaCl, 1mM EDTA) to...

Embodiment 2

[0045] In order to verify the specific detection of the magnetic microsphere DNA probe constructed by the present invention to the specific marker miRNA-21, according to the molecular sequence of miRNA-21, the molecules to be tested with different structures were selected, wherein M1, M2, M3 and miRNA- There are 1 or 2 different bases in 21, and the specific sequences of these six molecules are shown in Table 2.

[0046] Table 2 Sequences of six molecules with different structures

[0047] probe molecule Sequence (from 5' to 3' end) miRNA-21 (SEQ ID NO: 1) UAG CUU AUC AGA CUG AUG UUG A Mismatch 1 (M1) (SEQ ID NO: 6) UAG CUU AUC AGA C C G AUG UUG A

Mismatch 2 (M2) (SEQ ID NO: 7) UA C CUU AUC AGA C C G AUG UUG A

Mismatch 3 (M3) (SEQ ID NO: 8) UA C CUU AUC AGA C C G AUG UUG C

MiRNA-10b (SEQ ID NO: 9) UAC CCU GUA GAA CCG AAU UUG UG NC-MiRNA (NC) (SEQ ID NO: 10) UUG UAC UAC ACA AAA GUA CUG

[0048] The ma...

Embodiment 3

[0051] According to the operation steps of Example 1 and Example 2, real biological samples were selected, and the human serum containing miRNA-21 was diluted into three different concentrations of 10fM, 1pM, and 100pM, and the optimal experimental conditions were used to dilute the human serum in Example 1. The prepared magnetic microsphere DNA probe containing P1 was tested against different concentrations of human serum containing miRNA-21, and the test results are shown in Table 3. For 10fM, 1pM, and 100pM three kinds of test samples, the comparison of test results in serum and buffer solution shows that the RUF value has only a slight change, indicating that the magnetic microsphere DNA probe constructed by the present invention has great application in real human cancer diagnosis. potential.

[0052] Table 3 Test results of different concentrations of human serum containing miRNA-21

[0053] [miRNA], pM RFU%, buffer RFU%, serum Recovery% RSD% 0.01 ...

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Abstract

The invention relates to a construction method of a magnetic microsphere DNA probe for detecting miRNA molecules as well as a product and an application of the magnetic microsphere DNA probe, which belong to the technical field of molecular probes. The invention provides the magnetic microsphere DNA probe for detecting miRNA molecules, and the linear amplification of detection signals can be achieved on the premise that the number of initial miRNAs in a sample is kept unchanged. As the probe does not need PCR in the detection process, non-specific amplification is avoided, and the detection specificity is improved. Compared with a traditional separation method, the magnetic beads are used for separating complex components of a biochemical sample, separation and enrichment can be carried out at the same time, the separation speed and the enrichment efficiency are effectively improved, and meanwhile the sensitivity of analysis and detection is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of molecular probes, and in particular relates to a construction method of a magnetic microsphere DNA probe for detecting miRNA molecules, as well as products and applications thereof. Background technique [0002] Cancer, also called malignant tumor, has biological characteristics such as abnormal proliferation, uncontrolled growth, invasion and metastasis. It can destroy the structure and function of tissues and organs, cause necrosis, hemorrhage and infection, and the patient may eventually die due to organ failure. Currently, lung cancer remains the most life-threatening disease and is becoming more and more common worldwide due to its high morbidity and mortality. According to statistics released by the National Cancer Center, the number of new lung cancer patients in my country in 2015 was 787,000, accounting for about 20% of new cancer cases. To make matters worse, the number of new cases of lung ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6886C12N15/11
CPCC12Q1/6806C12Q1/6886C12Q2600/158C12Q2600/178C12Q2525/207C12Q2563/143C12Q2563/149C12Q2563/131C12Q2563/107
Inventor 查克·特里里何露·德杰比周大明穆罕默德·巴里孔祥东石彪田荣王德强
Owner CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
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