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Method for high-throughput detection of different target protein contents in plurality of samples to be detected and special kit of method

A protein content and kit technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of difficult protein molecules, difficult to prepare high-density microarrays, etc.

Pending Publication Date: 2020-05-12
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of this method is that it can be used as a quantitative detection method, and it ensures that the high-level structure of the protein can be detected; the disadvantage is that sometimes it is difficult to find a pair of protein molecules that can be connected to the same molecule and match, and this method It is difficult to prepare high-density microarrays, and it is very challenging to achieve high-throughput detection

Method used

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  • Method for high-throughput detection of different target protein contents in plurality of samples to be detected and special kit of method
  • Method for high-throughput detection of different target protein contents in plurality of samples to be detected and special kit of method
  • Method for high-throughput detection of different target protein contents in plurality of samples to be detected and special kit of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1. Preparation of a kit for detecting multi-sample multi-protein content by high-throughput sequencing

[0099] Based on PEA technology and DNA sequencing technology, the inventors of the present invention prepared a high-throughput sequencing kit for detecting multiple samples and multiple protein contents. The kit can simultaneously detect multiple proteins in multiple samples to be tested (ie, multiple samples), and the detection cost is low. The principle of the high-throughput sequencing of this kit to detect the content of multiple proteins in multiple samples is shown in figure 1 : figure 1 In A is the preparation process of the mixed probe. figure 1 In B, protein information is transformed into DNA information through reactions such as immunization, extension, and PCR amplification. figure 1 Middle C is the principle of high-throughput sequencing to detect multiple samples and multiple proteins.

[0100] The high-throughput sequencing kit for detecti...

Embodiment 2

[0133] Example 2, Preparation of a kit for detecting alpha-fetoprotein content by high-throughput sequencing

[0134] The inventors of the present invention prepared a kit for detecting the content of alpha-fetoprotein by high-throughput sequencing. The kit includes single-stranded DNA molecule A (hereinafter named ss-DNA1), alpha-fetoprotein antibody A (specifically, AFP capture antibody), single-stranded DNA molecule B (hereinafter named ss-DNA2), alpha-fetoprotein antibody B (Specifically AFP detection antibody), library construction PCR upstream primers, library construction PCR downstream primers (library construction PCR downstream primers 1, library construction PCR downstream primers 2, library construction PCR downstream primers 3 or library construction PCR downstream primers 4), DNA Standard product (aqueous solution of double-stranded specific DNA molecules), library construction PCR upstream primer-standard product, library construction PCR downstream primer-stand...

Embodiment 3

[0138] Example 3. Establishment of a method for detecting alpha-fetoprotein content using the kit prepared in Example 2

[0139] 1. Preparation of mixed probes

[0140] 1. Add DBCO-NHS and 30 μg AFP capture antibody to 30 μL pH7.2, 0.01 mM PBS buffer to obtain a reaction system; the concentration of DBCO-NHS in this reaction system is 8 nmol.

[0141] 2. Take the reaction system obtained in step 1, let it stand at 37°C for 30 minutes, and then ultrafiltration and centrifugation twice.

[0142] 3. After completing step 2, add ss-DNA1 to obtain a reaction system; in this reaction system, the concentration of ss-DNA1 is 0.5 nM.

[0143] 4. Take the reaction system obtained in step 3, let it stand at 37°C for 5 hours, and then ultrafiltration and centrifugation twice.

[0144] 5. After completing step 4, add ultrapure water to dilute to obtain AFP capture antibody-ss-DNA1 probe with a DNA concentration of 30 μg / mL.

[0145] 6. According to the above steps 1-5, replace the AFP c...

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Abstract

The invention discloses a method for high-throughput detection of different target protein contents in a plurality of samples to be detected and a special kit of the method. The inventor of the invention combines proximity extension assay (PEA) and the DNA sequencing technology to realize simultaneous detection of multi-protein content of various samples, and the specific method comprises the following steps: ss-DNA (protein bar codes) with different sequences are marked aiming at different types of antibodies, and protein types are distinguished according to different ss-DNA sequences; protein information is converted into DNA information by means of the PEA technology; a section of different Barcode marking sequences (sample bar codes) is added for distinguishing different samples when adatabase is established; and a sequencer is used to separately detect sample bar code sequences, protein bar code sequences and the number of the protein bar code sequences, so as to realize simultaneous detection of various protein contents of a plurality of samples. The method provided by the invention has an important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for high-throughput detection of different target protein contents in a plurality of samples to be tested and a special kit thereof. Background technique [0002] The methods for multi-protein trace detection mainly include Luminex technology, biorad bio-plex technology and protein chip technology. [0003] Luminex technology combines microspheres and flow cytometry, using color-coded microspheres to covalently cross-link monoclonal antibodies, and then adding fluorescein-labeled detection antibodies after binding to the target molecules to be determined, and then scanning the fluorescent codes by laser To identify individual microspheres and detect fluorescence intensity to determine the concentration of the analyte. Since different color-coded microspheres can be used to detect different proteins, multiple target molecules can be analyzed simultaneously in one...

Claims

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Application Information

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IPC IPC(8): C12Q1/6804C12Q1/6851
CPCC12Q1/6804C12Q1/6851C12Q2535/122C12Q2563/185C12Q2533/101C12Q2545/114
Inventor 赵芳李舟章文蔚刘二凯张长领陈奥
Owner SHENZHEN HUADA GENE INST
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