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A nucleic acid aptamer for glycosylated hemoglobin and its application and kit for detecting glycosylated hemoglobin

A technology of glycosylated hemoglobin and nucleic acid aptamer, applied in the field of biochemical detection, can solve problems such as poor affinity of glycosylated hemoglobin, unrecorded and public performance parameters, etc., and achieve high specificity and sensitivity

Active Publication Date: 2019-05-10
SINOCARE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this prior art, it does not record and disclose the specific performance parameters of its aptamer, such as affinity with glycosylated hemoglobin, detection limit, binding constant, etc., and can only be roughly speculated on the results of several results in Example 5. Analysis of its binding constant may be greater than 50nM, that is, it has poor affinity with glycosylated hemoglobin

Method used

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  • A nucleic acid aptamer for glycosylated hemoglobin and its application and kit for detecting glycosylated hemoglobin
  • A nucleic acid aptamer for glycosylated hemoglobin and its application and kit for detecting glycosylated hemoglobin
  • A nucleic acid aptamer for glycosylated hemoglobin and its application and kit for detecting glycosylated hemoglobin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Surface plasmon resonance method to detect the binding constant of the glycated hemoglobin nucleic acid aptamer and the glycated hemoglobin

[0025] Incubate and assemble 20 μg / ml glycosylated hemoglobin on the gold membrane, block with BSA and wash the unassembled protein, read the data, and then dissolve the nucleotide sequences shown in SEQ ID NO: 1 and 2 in the binding buffer The nucleic acid aptamer is added to the gold membrane, and after the reaction is completed, the unbound sequence is eluted, and the wavelength redshift data is read after the value is stable. According to this method, read the values ​​of the glycosylated hemoglobin nucleic acid aptamer concentrations of 6.25nM, 12.5nM, 25nM, 50nM, and 100nM, and then fit the nucleotide sequences shown in SEQ ID NO: 1 and 2 according to the values. The Kd value of the binding constant between the child and glycosylated hemoglobin (calculated by sigmaplot software). The wavelength redshift data are ...

Embodiment 2

[0037] Example 2: Antibody sandwich method to detect the binding of glycated hemoglobin nucleic acid aptamer to glycated hemoglobin

[0038]Take the glycosylated hemoglobin nucleic acid aptamer with the nucleotide sequences shown in SEQ ID NO: 1 and 2, use the antibody sandwich method to investigate the binding of the sequence to the glycosylated hemoglobin, use the 4 aptamers in CN102965378A as performance controls, and perform specific experiments The operation is as follows:

[0039] The glycosylated hemoglobin nucleic acid aptamer with the nucleotide sequences shown in SEQ ID NO: 1 and 2 is taken, and biotinylated glycosylated hemoglobin nucleic acid aptamer is obtained by labeling biotin at the 3' end and connecting 10 T bases. A layer of avidin was coated on the microtiter plate (coated avidin concentration 1 μg / mL, 100 μl per well), and after blocking, the biotinylated glycosylated hemoglobin nucleic acid aptamer (100 nM, 100 μl per well) was mixed with the coated Afte...

Embodiment 3

[0043] Example 3: Antibody sandwich method to detect the binding of glycosylated hemoglobin nucleic acid aptamer to hemoglobin

[0044] Take the glycosylated hemoglobin nucleic acid aptamer with the nucleotide sequences shown in SEQ ID NO: 1 and 2, and use the antibody sandwich method to investigate the binding of the sequence to hemoglobin. The specific test operation is as follows:

[0045] The glycosylated hemoglobin nucleic acid aptamer with the nucleotide sequences shown in SEQ ID NO: 1 and 2 is taken, and biotinylated glycosylated hemoglobin nucleic acid aptamer is obtained by labeling biotin at the 3' end and connecting 10 T bases. A layer of avidin was coated on the microtiter plate (coated avidin concentration 1 μg / mL, 100 μl per well), and after blocking, the biotinylated glycosylated hemoglobin nucleic acid aptamer (100 nM, 100 μl per well) was mixed with the coated After being incubated with avidin, different concentration gradients (1μg / mL, 1×10 -1 μg / mL, 1×10 -...

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Abstract

The invention relates to the technical field of biochemical detection and discloses a nucleic acid aptamer for glycosylated hemoglobin, an application thereof and a kit for detecting glycosylated hemoglobin. The nucleic acid aptamer provided by the invention has a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2 or has the nucleotide sequence under the condition of one or more situations of substituting basic group, missing basic group and adding basic group on the basis of the nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2. The invention provides two novel nucleic acid aptamers for glycosylated hemoglobin. The binding constant of the nucleic acid aptamer with the glycosylated hemoglobin can reach up to nM level; the combination with the glycosylated hemoglobin is less; the specificity and the sensitivity are ultrahigh; a signal gradient is obvious when a practical sample is detected; the nucleic acid aptamer is obviously different from the negative control; the nucleic acid aptamer can be applied to the detection research on the glycosylated hemoglobin content.

Description

technical field [0001] The invention relates to the technical field of biochemical detection, in particular to a nucleic acid aptamer for glycosylated hemoglobin and its application and a kit for detecting glycosylated hemoglobin. Background technique [0002] Glycosylated hemoglobin is a kind of glycoprotein, which was first separated from other types of hemoglobin in 1958. It is formed by combining blood sugar and hemoglobin through an irreversible reaction, and is proportional to blood sugar concentration. It can last for about 120 days and can reflect The situation of blood sugar in 8-12 weeks. The content of glycosylated hemoglobin in the blood has little interference with the time of blood drawing, whether it is fasting, whether insulin is used or not. The International Diabetes Federation has launched a new edition of the Asia-Pacific Diabetes Prevention and Control Guidelines, which clearly stipulates that glycosylated hemoglobin is the internationally recognized "g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/72
CPCC12N15/115C12N2310/16G01N33/723G01N2333/805
Inventor 刘振华
Owner SINOCARE