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Kit for simultaneously detecting multiple biomarkers based on nucleic acid rolling circle amplification reaction

A rolling circle amplification reaction and biomarker technology, applied in the field of marker detection, can solve the problems of inability to jointly detect multiple biomarkers at the same time, long reaction time, cumbersome operation, etc.

Pending Publication Date: 2021-03-26
湖南博奥瑞康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above common immunoassay techniques have their own advantages, but also have various defects, such as low sensitivity, long reaction time, cumbersome operation, and inability to jointly detect multiple biomarkers at the same time, etc.

Method used

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  • Kit for simultaneously detecting multiple biomarkers based on nucleic acid rolling circle amplification reaction
  • Kit for simultaneously detecting multiple biomarkers based on nucleic acid rolling circle amplification reaction
  • Kit for simultaneously detecting multiple biomarkers based on nucleic acid rolling circle amplification reaction

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preparation example Construction

[0034] The preparation methods of ssDNA1-cTnIAb1, ssDNA2-Myo Ab1 and ssDNA3-CKMB Ab1 in the specific antibody solution in component 1 of the present invention are the same, so only the preparation method of ssDNA1-cTnI Ab1 will be described in detail. The preparation method of ssDNA3-CKMB Ab1 will not be repeated here. The preparation method of ssDNA1-cTnIAb1 of the present invention preferably includes:

[0035] (1) Synthesize ssDNA1 with DIBO group modified at the 5' end to obtain DIBO-ssDNA13;

[0036] (2) carrying out diazotization treatment to cTnIAb1, so that the sugar chain on the antibody Fc fragment is modified to generate a diazo group to obtain N3-cTnIAb1;

[0037] (3) carry out coupling reaction with described DIBO-ssDNA1 and N3-cTnI Ab1, obtain described ssDNA1-cTnIAb1;

[0038] Step (1) and step (2) do not have a time sequence relationship.

[0039] The nucleotide sequence of ssDNA1 of the present invention is preferably shown in SEQ ID NO. 1: AAGTATTACCAGAAAC...

Embodiment 1

[0068] The preparation method of each component in the kit

[0069] 1. Preparation method of component 1:

[0070] 1.1 Preparation of ssDNA1-cTnIAb1

[0071] ①Synthesis of single-stranded DNA1 (DIBO-ssDNA1, SEQ ID NO.1) with modified DIBO group at the 5' end;

[0072] ②Using SiteClick of ThermoFisher TM Antibody Azido Modification Kit treats cTnIAb1, so that the sugar chain on the Fc fragment of the antibody is modified to form a diazo group;

[0073] 3. The single-stranded DNA1 (DIBO-ssDNA1) with the modified DIBO group at the 5' end is reacted and coupled with the diazotized cTnIAb1 (N3-cTnIAb1) to generate ssDNA1-cTnIAb1;

[0074] Table 1 Coupling system and reaction conditions

[0075]

[0076] ④ After the reaction, use molecular sieve to purify to obtain ssDNA1-cTnIAb1.

[0077] 1.2 Labeling method of ssDNA2-Myo Ab1

[0078] ① Synthesize single-stranded DNA2 with DIBO group modified at the 5' end (DIBO-ssDNA2, SEQ ID NO.2);

[0079] ②Using SiteClick of ThermoFi...

Embodiment 2

[0141] Sample testing and evaluation

[0142] 1. Sample Preparation

[0143] 1.1 cTnI sample preparation

[0144] The pure cTnI was prepared into samples with different concentration gradients with PBS buffer, the concentrations were 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 1, 10, 25, 50ng / ml;

[0145] 1.2 Myo sample preparation

[0146] The pure Myo product was prepared into samples with different concentration gradients with PBS buffer, the concentrations were 0.1, 1, 5, 10, 50, 100, 500, 1000ng / ml;

[0147] 1.3 CKMB sample preparation

[0148] Take pure CKMB and prepare samples with different concentration gradients with PBS buffer, the concentrations are respectively 0.1, 0.5, 1, 2, 5, 10, 20, 50, 100, 200ng / ml;

[0149] 1.4 Preparation of cTnI, Myo, CKMB composite serum samples

[0150] A total of 20 samples from patients with myocardial infarction in the Department of Cardiology and healthy samples for physical examination were taken, and hs-cTnI, Myo, and CKMB were d...

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Abstract

The invention provides a kit for simultaneously detecting multiple biomarkers based on nucleic acid rolling circle amplification reaction, and relates to the technical field of marker detection. The kit provided by the invention comprises four independent components: a specific antibody solution for labeling single-stranded deoxyribonucleic acid; a magnetic bead suspension coated with a specific antibody of the biomarker; a reaction solution containing cyclic deoxyribonucleic acid; and reaction solutions containing different probes. When the kit disclosed by the invention is used for detectingvarious markers, the detection result is similar to the detection result of Japonica chemiluminiscence, even lower concentration (ag / ml) can be detected within enough time, and the sensitivity is obviously superior to that of a chemiluminiscence detection method. For example, when cardiovascular disease biomarkers such as cardiac troponin I (cTnI), myoglobin (Myo) and creatine kinase isozyme (CKMB) are detected, detection of three indexes is completed in one reaction cup at the same time, and results are not interfered with one another.

Description

technical field [0001] The invention belongs to the technical field of marker detection, and in particular relates to a kit for simultaneous detection of multiple biomarkers based on a nucleic acid rolling circle amplification reaction. Background technique [0002] Immunodetection technology is one of the most commonly used and classic detection technologies at present, and its main features are: The specific recognition of antibodies and antigens is used to combine with each other to generate an immune binding reaction. According to the different markers, there are mainly radioimmunoassay technology, enzyme-linked immunosorbent assay technology, latex-enhanced immunoturbidimetric detection technology, immunochromatographic detection technology and chemiluminescence detection technology. Radioimmunoassay technology is based on the antigen-antibody reaction that uses radionuclides as markers to label antigens or antibodies. Radioimmunoassays require special equipment and hav...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/573G01N33/58C12Q1/6804
CPCG01N33/6893G01N33/6887G01N33/573G01N33/58C12Q1/6804G01N2333/4712G01N2333/805G01N2333/9123G01N2800/32C12Q2531/125C12Q2563/107C12Q2521/301Y02A50/30
Inventor 严竹林
Owner 湖南博奥瑞康生物科技有限公司
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