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Detection method and kit for thrombin based on double Aptamer sandwich structure open ring mediated isothermal amplification

A sandwich structure and detection method technology, applied in the field of biological detection, can solve the problems of narrow applicability and achieve the effect of not relying on large equipment, high sensitivity and good specificity

Active Publication Date: 2018-03-13
HUNAN INSTITUTE OF ENGINEERING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Loop-mediated isothermal amplification is mainly used for the detection of microorganisms in biology. The mechanism is to detect their nucleic acids. This method can only detect nucleic acids and has a narrow range of applicability.

Method used

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  • Detection method and kit for thrombin based on double Aptamer sandwich structure open ring mediated isothermal amplification
  • Detection method and kit for thrombin based on double Aptamer sandwich structure open ring mediated isothermal amplification
  • Detection method and kit for thrombin based on double Aptamer sandwich structure open ring mediated isothermal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Probe Design

[0073] (1) The structure of P1 is as follows (5'-3'): (SEQ ID NO.1)

[0074]

[0075] Among them, the underline is the extended chain, the bold is the thrombin recognition sequence, and the 5' end is modified with biotin molecules.

[0076] (2) The structure of P2 is as follows (5'-3'): (SEQ ID NO.2)

[0077]

[0078] The bold font is the thrombin recognition sequence; the underlined region in the middle is completely complementary to the 3' free end of the primary basic structure of LAMP, and forms a hybrid dimer after combining; the italic font is the free end.

[0079] (3) The structure of P3-1 is as follows (5'-3'): SEQ ID NO.3

[0080]

[0081] Among them, the underlined part is the intramolecular hybridization complementary region, and the black bold part at the 3' end is the connection complementary region, which is complementary to the P3-COM probe.

[0082] (4) The structure of P3-2 is as follows (5'-3'): SEQ ID NO.4

[0083]

[0...

Embodiment 2

[0102] Detection of different concentrations of thrombin

[0103] 1. Microsphere functionalization

[0104] Take 100uL of avidin-modified microspheres (1% or 10mg / ml) in a 1.5ml EP tube, centrifuge (3500rpm, 5min, the following conditions are used for centrifugation in subsequent steps), and remove the supernatant. Wash 3 times with 100 uL Binding / wash buffer (20 mM Tris-HCl, pH=7.5, 1 M NaCl, 1 mM EDTA, 0.0005% Triton X-100), centrifuge, and remove the supernatant. The microspheres were resuspended in 20uL Binding / Wash buffer, 4.5uL of 10uM biotinylated capture probe P1 was added, and 25.5uL Binding / Wash buffer was added to a final volume of 50uL. Incubate at room temperature for 30 min, agitate with a pipette every 5 min. Wash three times with 100uL Binding / wash buffer, and centrifuge to remove the supernatant. Add 100uL 10% BSA TB buffer (0.1g BSA, dissolved in 1ml TB buffer solution, TB buffer: 20mM Tris-HCl, pH 7.4, 140mMNaCl, 5mM KCl, 1mM MgCl 2 , 1mM CaCl 2 ) at 37...

Embodiment 3

[0115] Anti-interference ability analysis

[0116] In order to test the selectivity of the system of the present invention for detection of thrombin, four other interfering molecules (BSA, IgG, Lysozyme, ATP) were selected, and their degree of interference to the detection of thrombin was tested respectively. Among them, the concentration of thrombin was 5pg / mL, and the concentration of other interfering substances was 5ng / mL.

[0117] see results Figure 4 . From the results, although the concentration of interfering molecules is 1000 times that of thrombin, the detection signal of thrombin is far greater than the detection signals of other interfering molecules, which shows that the method system of the present invention has a high selectivity for thrombin sex.

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Abstract

The invention relates to a detection method and a kit for thrombin based on double Aptamer sandwich structure open ring mediated isothermal amplification. The detection method comprises the followingsteps: mixing a double Aptamer sandwich structure with a LAMP (Loop-Mediated Isothermal Amplification) primary base structure; forming an LAMP base structure under the action of Nb.BsrDI incision enzyme; carrying out LAMP under the action of primers; adding heme in an amplified product; detecting the thrombin by using an ABTS-H2O2 reaction, wherein the 3' terminal of the double Aptamer sandwich structure is provided with a free single-chain sequence, and initiating the LAMP. The invention also provides a kit for the method. The detection method disclosed by the invention first combines a double Aptamer sandwich detection method, an LAMP technology and a G-quadruplex-heme DNA enzyme enzymatic reaction and is a novel detection method with high sensitivity and high specificity for the thrombin.

Description

technical field [0001] The invention relates to the technical field of biological detection, and relates to a thrombin detection method and a kit based on a ring-mediated isothermal amplification signal amplification technology and a nucleic acid aptamer recognition technology. Background technique [0002] Thrombin is a serine protease and the main effector protease in the blood coagulation cascade, exhibiting procoagulant and anticoagulant properties. When circulating coagulation factors come into contact with tissue factor in exposed extravascular tissue, thrombin accumulates on the tissue. Thrombin not only participates in the blood coagulation process, but also acts as an important extracellular signaling molecule. By activating thrombin receptors, it participates in a series of pathological processes, such as: cleaving fibrinogen to form fibrin thrombus and activating platelets. Therefore, the development of highly sensitive thrombin detection methods with practical v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/56
CPCC12Q1/56C12Q1/6844G01N2333/974C12Q2531/119C12Q2525/205C12Q2563/149
Inventor 张何蒋序春傅昕
Owner HUNAN INSTITUTE OF ENGINEERING
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