Exosome detecting method based on adapter and rolling ring amplification

A technology of rolling circle amplification and detection method, which is applied in measuring devices, material analysis through electromagnetic means, instruments, etc., can solve the problems of low sensitivity, high background signal, detection interference, etc., and achieve the effect of improving detection sensitivity

Inactive Publication Date: 2019-04-19
NANJING DRUM TOWER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention solves the problem of low sensitivity in the method of using exosome-specific antibodies or aptamers as

Method used

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  • Exosome detecting method based on adapter and rolling ring amplification
  • Exosome detecting method based on adapter and rolling ring amplification
  • Exosome detecting method based on adapter and rolling ring amplification

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0031] Example 1: Preparation of circular template and rolling circle amplification products.

[0032] 1. The phosphorylated padlock probe (100μM) and 1μL ligation probe (100μM) were hybridized in T4 ligase buffer; 60U T4 DNA ligase was added, and the mixture was incubated at 16°C overnight; T4 ligase was kept at 65°C Inactivate in 15 minutes, add 20U exonuclease I and 50U exonuclease III to digest the remaining single-stranded DNA and double-stranded DNA; the enzyme is denatured by heating at 85°C for 10 minutes.

[0033] The sequence of padlock probe and connection probe is as follows:

[0034] Padlock probe:

[0035] 5’-TAGCACGGACATTTTCCCAACCCGCCCTACCCTTTTTTTTTTCCCAACCCGCCCTACCCTTTTGTAACTGTTTCCTTC-3’

[0036] Connection probe: 5’-TGTCCGTGCTAGAAGGAAACAGTTAC-3’

[0037] 2. Add 1μL phi29DNA polymerase buffer, 0.5μL 10mM dNTPs, 0.2μL 0.1mg / mL BSA, 10U phi29DNA polymerase, 10μL 100nM aptamer-primer probe and 10μL 120nM circular template, and incubate the electrode at 37℃ 2 hours.

[0038]...

Example Embodiment

[0041] Example 2: Antibodies are modified on the gold electrode surface and used to capture gastric cancer exosomes and start rolling circle amplification.

[0042] 1. The gold electrode is polished with 0.05mm alumina slurry, the electrode is rinsed with ultrapure water, and dried at room temperature; the pretreated gold electrode is soaked in a PBS solution containing 15mM cysteamine overnight to produce self-contained cysteamine Assemble the monolayer; soak the gold electrode in a 5% glutaraldehyde aqueous solution and react at 37°C for 6 hours; incubate the gold electrode with 10μL of 20μg / mL anti-CD63 antibody for 1 hour at 37°C; soak the electrode in 5mg / mL Incubate in BSA solution for 1 hour at 37°C and rinse with ultrapure water.

[0043] 2. Incubate the antibody-modified electrode with the gastric cancer exosome solution resuspended in PBS for 1 hour at 37°C, and wash the electrode with PBS solution; modify 10μL of 100nM aptamer-primer probe on the electrode surface and in...

Example Embodiment

[0045] Example 3: Electrode working process

[0046] 1. Preparation of gold electrode with modified antibody: (1) The gold electrode is polished with 0.05mm alumina slurry, the electrode is rinsed with ultrapure water, and dried at room temperature; (2) The pretreated gold electrode is prepared with 15mM cysteamine Soak in PBS solution overnight to produce a self-assembled monolayer of cysteamine; (3) Soak the gold electrode in a 5% glutaraldehyde aqueous solution and react at 37°C for 6 hours; (4) The gold electrode and 10μL 20μg / mL The anti-CD63 antibody was incubated at 37°C for 1 hour; (5) The electrode was immersed in a 5mg / mL BSA solution, incubated at 37°C for 1 hour, and washed with ultrapure water.

[0047] 2. Preparation of circular template: (1) Phosphorylated padlock probe (100μM) and 1μL ligation probe (100μM) were hybridized in T4 ligase buffer; (2) 60U T4 DNA ligase was added, and the mixture was mixed in 16 Incubate overnight at ℃; (3) Keep T4 ligase at 65℃ for 15 ...

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Abstract

The invention relates to an exosome detecting method based on adapter and rolling ring amplification. An electrochemical sensor method is applied for constructing an adapter probe based on exosome singularity. A signal is generated by means of H2O2 reaction catalysis by G-tetrad-Hemin simulated peroxidase. A large number of G-tetrads are synthesized through rolling ring amplification for performing signal amplification. The method realizes quantitative detection for the exosomes and is used for detecting an actual serum sample. According to the method of the invention, the adapter probe basedon the exosome singularity is constructed through applying an electrochemical sensor method; the signal is generated through H2O2 catalysis by the G-tetrad-Hemin simulated peroxidase; and furthermorea large number of G-tetrads are synthesized through rolling ring amplification for realizing signal amplification, thereby greatly improving detecting sensitivity of the exosome.

Description

technical field [0001] The invention relates to the fields of biochemical detection and molecular diagnosis, in particular to an exosome detection method based on aptamers and rolling circle amplification. Background technique [0002] Exosomes are bilayer membranous vesicles secreted by various types of cells, with a diameter of 30-100 nm, which contain cell-specific proteins, lipids, nucleic acids and other components. Exosomes can be effectively detected in almost all types of human body fluids, including peripheral blood, saliva, urine, etc. The important signaling molecules carried and transmitted by exosomes form a new intercellular information transmission system, which affects the physiological state of cells and is closely related to the occurrence and process of various diseases. For example, exosomes secreted by tumor cells can enter Capillaries in the lymphatic system and tumor tissue play a role in the regulation of malignant tumors. In addition, the character...

Claims

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Application Information

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IPC IPC(8): G01N27/327
CPCG01N27/3275
Inventor 李智洋何农跃黄蓉蓉
Owner NANJING DRUM TOWER HOSPITAL
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