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Carcinoembryonic antigen detection kit based on nucleic acid aptamer autocatalysis effect, and preparation method thereof

A detection kit, a technology for carcinoembryonic antigen, applied in the field of tumor marker detection, can solve the problems of chemical analysis method, fluorescence analysis method, electrochemiluminescence analysis method, surface-enhanced Roman scattering method, surface-enhanced plasmon resonance method, etc. , to achieve the effect of convenient storage of reagents, low cost and simple method

Active Publication Date: 2017-08-18
菏泽医学专科学校
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, important progress has been made in the application of aptamers in the detection and analysis of tumor markers in blood samples, mainly focusing on electrochemical analysis, fluorescence analysis, electrochemiluminescence analysis, surface-enhanced Roman scattering, surface-enhanced Plasma resonance method, etc. have greatly improved in terms of sensitivity and selectivity, but still have high requirements for detection instruments and experimental techniques.

Method used

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  • Carcinoembryonic antigen detection kit based on nucleic acid aptamer autocatalysis effect, and preparation method thereof
  • Carcinoembryonic antigen detection kit based on nucleic acid aptamer autocatalysis effect, and preparation method thereof
  • Carcinoembryonic antigen detection kit based on nucleic acid aptamer autocatalysis effect, and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0058] A carcinoembryonic antigen detection kit based on nucleic acid aptamer autocatalysis, comprising:

[0059] The first aptamer consists of a 3' end of a nucleotide fragment with a nucleotide sequence as shown in SEQ ID NO.1 bound to a biotin through a hydroxyl group;

[0060] The first aptamer sequence is 5'-TTAACTTATTCGACCATATTTTTTTTTT-biotin-3';

[0061] The second aptamer is composed of a nucleotide fragment whose nucleotide sequence is shown in SEQ ID NO.2;

[0062] The second aptamer sequence is 5'-CCCATAGGGAAGTGGGGGA-3';

[0063] Streptavidin-coated magnetic beads with a concentration of 4 mg / mL, the dosage is 4 μg;

[0064] 0.2mol / L KCl solution, 20μL;

[0065] 10 μmol / L hemin solution, 1 μL;

[0066] The reaction buffer is a phosphate buffer solution with pH 7.4, the chromogenic solution is 0.1mg / mL 3,3',5,5'-tetramethylbenzidine (TMB) solution, and the stop reaction solution is a concentration of 2mol / L H 2 SO 4 solution.

[0067] The above-mentioned prep...

Embodiment 2

[0079] The application of the above-mentioned carcinoembryonic antigen detection kit based on nucleic acid aptamer autocatalysis in the detection of carcinoembryonic antigen, the specific steps are as follows:

[0080] 1. Preparation of serum samples to be tested: collect 3ml of whole blood with a vacuum coagulation tube, let it stand for 1 hour, rotate at 3000 rpm, centrifuge for 10 minutes, and take 10 μL of the supernatant to be tested.

[0081] The buffer solution used in the experiment was PO 4 3- The concentration is 0.01mol / L pH7.4 phosphate (PBS) buffer solution.

[0082] 2. Preparation of magnetic beads functionalized with the first aptamer of carcinoembryonic antigen:

[0083] In the kit prepared in Example 1, 30 μL of pH7.4 phosphate (PBS) buffer solution, 1 μL of 4 mg / mL streptavidin-coated magnetic beads with a diameter of 1 μm, and the reaction buffer for the first aptamer (0.01mol / L, pH7.4 phosphate buffer solution) diluted to 100μmol / L, fully shaken for 10 m...

Embodiment 3

[0100] A carcinoembryonic antigen detection kit based on nucleic acid aptamer autocatalysis, comprising:

[0101] The first aptamer consists of a 3' end of a nucleotide fragment with a nucleotide sequence as shown in SEQ ID NO.1 bound to a biotin through a hydroxyl group;

[0102] The first aptamer sequence is 5'-TTAACTTATTCGACCATATTTTTTTTTT-biotin-3';

[0103] The second aptamer is composed of a nucleotide fragment whose nucleotide sequence is shown in SEQ ID NO.2;

[0104] The second aptamer sequence is 5'-CCCATAGGGAAGTGGGGGA-3';

[0105] Streptavidin-coated magnetic beads with a concentration of 10 mg / mL, the dosage is 10 μg;

[0106] 1.0mol / L KCl solution, 10μL;

[0107] 5 μmol / L hemin solution, 5 μL;

[0108] The reaction buffer is a phosphate buffer solution with pH 7.2, the chromogenic solution is 3,3',5,5'-tetramethylbenzidine (TMB) solution with a concentration of 0.1mg / mL, and the stop reaction solution is a concentration of 1.0mol / L of H 2 SO 4 solution.

...

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Abstract

The present invention relates to a carcinoembryonic antigen detection kit based on a nucleic acid aptamer autocatalysis effect, and a preparation method thereof. The carcinoembryonic antigen detection kit comprises: a first aptamer, wherein a nucleotide segment having a nucleotide sequence represented by SEQ ID NO.1 is combined with a biotin at the 3' terminal through hydroxy to form the first aptamer; a second aptamer, wherein the second aptamer comprises a nucleotide segment having a nucleotide sequence represented by SEQ ID NO.2; streptavidin-coated magnetic beads; a KCl solution; a hemin solution; a reaction buffer liquid; a coloring solution; and a reaction terminating solution. According to the present invention, the kit detects carcinoembryonic antigens, and does not require animal immunization antibody and enzyme labeling process compared to the existing detection method; the nucleic acid aptamer is chemically synthesized, and has good stability and good environmental tolerance; the reagents are convenient to store; and the quantitative detection can be performed only with the one visible spectrophotometer.

Description

technical field [0001] The invention relates to a carcinoembryonic antigen detection kit based on nucleic acid aptamer autocatalysis and a preparation method thereof, in particular to a magnetic bead separation technology combined with nucleic acid aptamer specific capture and autocatalysis for colorimetric detection of carcinoembryonic antigen The kit belongs to the technical field of tumor marker detection. Background technique [0002] Cancer is one of the persistent diseases in the field of medicine today, which seriously threatens human health. With the deterioration of human living environment, this threat is on the rise. According to the statistics of the World Health Organization, the cure rate of early cancer can reach 80%, but most malignant tumors lack specific clinical manifestations in the early stage of the disease, and the sensitivity of traditional clinical diagnostic methods such as imaging and cytology is not high enough Moreover, precision diagnostic inst...

Claims

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Application Information

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IPC IPC(8): G01N33/574
CPCG01N33/57473
Inventor 邹明静
Owner 菏泽医学专科学校
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