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Method for visually detecting nucleic acid

A detection method and nucleic acid technology, which are applied in the directions of biochemical equipment and methods, and the determination/inspection of microorganisms, can solve the problems of easy inactivation of enzymes, difficult preservation, and high consumption, and achieve low cost, simple reaction, and biological safety. high effect

Inactive Publication Date: 2019-03-19
SHANGHAI NAT ENG RES CENT FORNANOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, enzymes are easily inactivated, difficult to store, and costly, so non-enzyme-involved amplification reactions have become research hotspots

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] A nucleic acid visualization detection method, specifically as follows:

[0017] (1) Streptavidin-modified magnetic beads linked to biotin-modified probes:

[0018] Take 100 μL streptavidin-modified magnetic beads (SA-beads) and place them on the magnetic stand, remove the supernatant after 1 min, and use 500 μL buffer I (10 mM Tris-HCl (pH=7.5) for precipitation, 1 mM EDTA, 1M NaCl). Afterwards, 50 μL of 100 μM biotin-modified probe (DNA 1) was mixed thoroughly with SA-beads, and reacted at room temperature for 30 min. Place the mixture on a magnetic stand, remove the supernatant by magnetic separation, wash the magnetic beads three times with buffer I, resuspend the pellet in 100 μL of phosphate buffer (PBS, pH=7.4), and record it as beads-DNA 1.

[0019] (2) Gold nanoparticles linked to sulfhydryl-modified probes:

[0020] Take 5 μL of 100 μM thiol-modified probe (DNA 2) and add 400 μL of 18 nm Au NPs (0.25 nM) to incubate at room temperature for 6 hr. Add 5 μL o...

Embodiment 2

[0024] A nucleic acid visualization detection method, specifically as follows:

[0025] (1) Streptavidin-modified magnetic beads linked to biotin-modified probes:

[0026] Take 100 μL streptavidin-modified magnetic beads (SA-beads) and place them on the magnetic stand, remove the supernatant after 1 min, and use 500 μL buffer I (10 mM Tris-HCl (pH=7.5) for precipitation, 1 mM EDTA, 1M NaCl). Afterwards, 50 μL of 100 μM biotin-modified probe (DNA 4) was mixed well with SA-beads, and reacted at room temperature for 30 min. Place the mixture on a magnetic stand, remove the supernatant by magnetic separation, wash the magnetic beads three times with buffer I, resuspend the pellet in 100 μL of phosphate buffer (PBS, pH=7.4), and record it as beads-DNA 4.

[0027] (2) Gold nanoparticles linked to sulfhydryl-modified probes:

[0028] Take 5 μL of 100 μM thiol-modified probe (DNA 5) and add 400 μL of 18 nm Au NPs (0.25 nM) to incubate at room temperature for 6 hr. Add 5 μL of 200 ...

Embodiment 3

[0032] A nucleic acid visualization detection method, specifically as follows:

[0033] (1) Streptavidin-modified magnetic beads linked to biotin-modified probes:

[0034] Take 100 μL streptavidin-modified magnetic beads (SA-beads) and place them on the magnetic stand, remove the supernatant after 1 min, and use 500 μL buffer I (10 mM Tris-HCl (pH=7.5) for precipitation, 1 mM EDTA, 1M NaCl). Afterwards, 50 μL of 100 μM biotin-modified probe (DNA 7) was mixed well with SA-beads, and reacted at room temperature for 30 min. Put the mixture on a magnetic stand, remove the supernatant by magnetic separation, wash the magnetic beads three times with buffer I, resuspend the pellet in 100 μL of phosphate buffer (PBS, pH=7.4), and record it as beads-DNA 7.

[0035] (2) Gold nanoparticles linked to sulfhydryl-modified probes:

[0036] Take 5 μL of 100 μM thiol-modified probe (DNA 8) and add 400 μL of 30 nm Au NPs (0.25 nM) to incubate at room temperature for 6 hr. Add 5 μL of 200 mM...

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Abstract

The invention relates to a method for visually detecting nucleic acid, which is a method by adopting nucleic acid as target and connecting magnetic beads and gold nano-particles by chemical self-connection reaction. According to the method, the reaction condition is controlled, a connecting probe complementary with the target nucleic acid part is respectively modified on surfaces of magnetic beadsand gold nano-particles, and the magnetic beads and gold nano-particles are connected by a self-connection reaction after matching with the target nucleic acid; the gold nano-particles which do not react are removed through magnetic separation, and visual detection on nucleic acid can be carried out through magnetic separation. The separation method is convenient, and materials are partially commercialized with high biosafety.

Description

technical field [0001] The invention relates to a method for visually detecting nucleic acid, in particular to a method for visually detecting nucleic acid by using a chemical self-ligation reaction to connect magnetic beads and gold nanoparticles. The invention belongs to the field of nanometer biological detection. Background technique [0002] Rapid detection and imaging of nucleic acids has great potential in biomedical applications. DNA and RNA contain important information in the pathological characteristics and development of diseases, and can be used as important biomarkers for studying cancer pathogenesis, identifying infection and drug resistance. In particular, these short noncoding nucleotides, microRNAs (miRNAs), have been found to regulate a range of fundamental cellular functions, such as cell proliferation, differentiation, and death. Studies have confirmed that the abnormal expression level of miRNAs is closely related to different diseases, such as cancer...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2563/137C12Q2563/143C12Q2563/149
Inventor 何丹农徐艳张兆坤陈玮嘉王萍金彩虹张芳
Owner SHANGHAI NAT ENG RES CENT FORNANOTECH
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