Thrombin detection method based on aptamer and telomerase amplification

A nucleic acid aptamer and detection method technology, applied in biochemical equipment and methods, microorganism determination/inspection, etc., can solve the problems of difficult thrombin determination, small signal variation range, narrow linear range, etc. Low cost, simple response effect

Pending Publication Date: 2019-09-20
SHANGHAI NAT ENG RES CENT FORNANOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is limited by the change of fluorescence intensity after the direct combination of nucleic acid aptamer and thrombin, the range of signal change is small, the linear range is narrow, and there is a certain deviation in the determination of fluorescence polarization for heterogeneous solutions. It is difficult to measure low concentration of thrombin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Streptavidin-modified magnetic beads linked to biotin-modified probes:

[0024]Take 10 μL of 100 μM biotin-modified DNA 1 and incubate at 90°C for 5 min, then slowly cool down to room temperature, so that the probe sequence forms a hairpin structure, which is designated as hairpin-DNA. 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, 1 mM EDTA, 1M Na Cl, pH=7.5) to resuspend. After 10 μL of 100 μM hairpin-DNA and 10 μL of 100 μM DNA 3 were mixed uniformly at a molar ratio of 3:2, they were thoroughly mixed with SA-beads and reacted at room temperature for 30 min. Place the mixture on a magnetic stand, remove unbound DNA by magnetic separation, wash the magnetic beads three times with buffer I, and use 100 μL of Tb buffer (22 mM Tris-HCl, 120 mM NaCl, 50 mM KCl, 0.85 mM CaCl 2 , 6 mM MgCl 2 , 6.8% glycerol, pH 7.8) resuspended to obtain mag...

Embodiment 2

[0032] 1. Streptavidin-modified magnetic beads linked to biotin-modified probes:

[0033] Take 10 μL of 100 μM biotin-modified DNA 2 and incubate at 90°C for 5 min, then slowly cool down to room temperature, so that the probe sequence forms a hairpin structure, which is designated as hairpin-DNA. 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, 1 mM EDTA, 1M Na Cl, pH=7.5) to resuspend. After 10 μL of 100 μM hairpin-DNA and 5 μL of 100 μM DNA 3 were mixed uniformly at a molar ratio of 2:1, they were thoroughly mixed with SA-beads and reacted at room temperature for 30 min. Place the mixture on a magnetic stand, remove unbound DNA by magnetic separation, wash the magnetic beads three times with buffer I, and use 100 μL of Tb buffer (22 mM Tris-HCl, 120 mM NaCl, 50 mM KCl, 0.85 mM CaCl 2 , 6 mM MgCl 2 , 6.8% glycerol, pH 7.8) resuspended to obtain mag...

Embodiment 3

[0041] 1. Streptavidin-modified magnetic beads linked to biotin-modified probes:

[0042] Take 10 μL of 100 μM biotin-modified DNA 1 and incubate at 90°C for 5 min, then slowly cool down to room temperature, so that the probe sequence forms a hairpin structure, which is designated as hairpin-DNA. 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, 1 mM EDTA, 1M Na Cl, pH=7.5) to resuspend. After 10 μL of 100 μM hairpin-DNA and 5 μL of 100 μM DNA 3 were mixed uniformly at a molar ratio of 2:1, they were thoroughly mixed with SA-beads and reacted at room temperature for 30 min. Place the mixture on a magnetic stand, remove unbound DNA by magnetic separation, wash the magnetic beads three times with buffer I, and use 100 μL of Tb buffer (22 mM Tris-HCl, 120 mM NaCl, 50 mM KCl, 0.85 mM CaCl 2 , 6 mM MgCl 2 , 6.8% glycerol, pH 7.8) resuspended to obtain mag...

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PUM

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Abstract

The invention relates to a thrombin detection method based on aptamer and telomerase amplification. The method has the advantages that the method targets thrombin, aptamer is used to identify the target, the signal is further amplified through telomerase amplification, and visual thrombin detection is achieved by further using magnetic separation; the method controls reaction conditions, the surface of a magnetic bead is modified with a probe for identifying thrombin, the probe comprises a thrombin aptamer sequence and a telomerase identification sequence, and the telomerase identification sequence at the terminal of the probe is exposed after the probe is combined with the target; the separation method is convenient, all materials are commercially available, and high bio-safety is achieved; the method is simple in reaction, low in cost and sensitive and visual in detection.

Description

technical field [0001] The invention relates to a thrombin detection method based on nucleic acid aptamer and telomerase amplification, a detection method of thrombin content, in particular to a nucleic acid aptamer captures thrombin and exposes a terminal telomerase recognition primer. Using telomerase to identify primers and perform nucleic acid amplification to obtain repeat products rich in guanine (G), which are combined with hematin for visual inspection. The invention belongs to the field of nanometer biological detection. Background technique [0002] The coagulation and anticoagulation mechanisms in normal humans maintain a dynamic balance. However, the generation of tumor tissue, infiltration and metastasis to surrounding tissues, and changes in the blood components of tumor patients will affect this balance, resulting in changes in the coagulation mechanism of patients. , resulting in varying degrees of bleeding tendency. Studies have shown that there is a corre...

Claims

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Application Information

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IPC IPC(8): C12Q1/682C12Q1/56
CPCC12Q1/682C12Q1/56C12Q2521/113C12Q2525/205C12Q2525/301
Inventor 徐艳张兆坤陈玮嘉朱君王萍
Owner SHANGHAI NAT ENG RES CENT FORNANOTECH
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