Tumor antigen expression examination primer and kit based on high-throughput sequencing method

A technique for detecting tumor antigens and primers, which is applied in the biological field and can solve problems such as low throughput and limited detection range

Active Publication Date: 2020-06-05
BEIJING IMMUPEUTICS MEDICINE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Viral antigens, cancer-testis antigens, tissue-specific tumor antigens, and tumor overexpressed antigens currently have relatively mature detection and identification methods at the protein level, mainly based on enzyme-linked immunosorbent assay (ELISA), antibody chips, etc. The throughput of these methods Antigens with low detection range limited by pre-existing antibodies

Method used

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  • Tumor antigen expression examination primer and kit based on high-throughput sequencing method
  • Tumor antigen expression examination primer and kit based on high-throughput sequencing method
  • Tumor antigen expression examination primer and kit based on high-throughput sequencing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] Example 1 Identification of Individualized Tumor Antigen Peptide Library Establishment

[0153] The workflow for identifying individualized tumor antigen peptide libraries using fresh tumor tissue or FFPE sections is as follows: figure 1 shown.

[0154] 1. Total RNA Extraction

[0155] 1) Use RNA extraction kit or DNA and RNA co-extraction kit to extract total RNA.

[0156] 2) Use Qubit TM RNA HS Assay Kit for total RNA quantification.

[0157] 3) Agilent RNA 6000 Pico Kit was used to determine the integrity of RNA.

[0158] 4) At least 10 ng of DNase-treated total RNA (≥1.43 ng / μL) is required for each reverse transcription reaction. For samples with low extraction concentration (concentration lower than 1.43ng / μL), take the largest volume (7μL) for reverse transcription, and the initial amount of library construction is <10ng, which belongs to high-risk library construction.

[0159] 5) For samples with DV 200%30% of samples, use 10-50ng input for reverse transc...

Embodiment 2

[0250] The different RNA starting amount of embodiment 2FFPE samples and repetition in batch

[0251] One case of FFPE sample with good RNA extraction quality was selected (RIN value 2.1, DV200 80, Figure 2A ), using 10ng, 20ng, and 50ng of total RNA as the starting amount, perform multiplex PCR amplification of the target sequence, build a library, and sequence it. It can be seen that when the total RNA quality is good, the total RNA can be as low as 10ng A better quality library was obtained ( Figure 2B -D). Each initial amount was repeated 3 times for library construction, and a total of 9 libraries were obtained with 3 initial amounts, and the sequencing was completed in the same batch. After obtaining the expression level (nrpm) of the target sequence in each library, Pearson correlation analysis was performed on the paired libraries. The correlation coefficient R was above 0.9, and the consistency was very high (see Figure 2E ).

Embodiment 3

[0252] Repeatability between batches of embodiment 3 fresh tumor cell samples

[0253] One fresh sample was selected, and under the same experimental conditions, the same initial amount of library construction (10ng) was used to construct the library, and 3 replicate libraries were obtained, which were repeated 2 times on the machine, and a total of 6 sequencing data were obtained. The expression level (nrpm) of the target sequence in each library was obtained, and the Pearson correlation analysis was carried out twice for the three libraries. The correlation coefficients between the two repetitions were all 0.99 or above, and the consistency was extremely high (see image 3 ).

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Abstract

The invention belongs to the technical field of biology and particularly relates to a tumor antigen expression examination primer and kit based on a high-throughput sequencing method and application of the tumor antigen expression examination primer and kit. The kit comprises plural pairs of amplification primers of a plurality of tumor antigens of Chinese common tumors (including lung cancer, liver cancer, pancreatic cancer, colorectum cancer and the like), and a reagent for RNA reverse transcription required in high-throughput sequencing, library construction and sequencing. The primer and kit can be used for performing once examination and analysis on a sample, can complete RNA expression level examination of 58, and has the characteristics of high throughput, high sensitivity, low examination list, high specificity and the kit.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer and a kit for detecting tumor antigen expression based on a high-throughput sequencing method. Background technique [0002] Tumor antigen refers to an immunogenic substance produced by tumor cells, which can stimulate the immune response of the host body. Due to the body's self-tolerance, protein substances normally present in the body are not antigenic. However, proteins that have not been immune-tolerant will trigger an immune response. Such proteins can be normal proteins that exist in the body, including cryptic antigens that are sequestered from the immune system, proteins that exist in very low amounts, or exist only at specific developmental stages proteins, or proteins with structural abnormalities due to mutations. Tumor antigens are usually derived from viral proteins, or structurally abnormal proteins caused by mutations, or normal proteins that are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6806C12Q1/6869C12N15/11C40B50/06
CPCC12Q1/6886C12Q1/6806C12Q1/6869C40B50/06C12Q2600/158C12Q2600/16C12Q2531/113C12Q2537/143C12Q2525/191C12Q2535/122
Inventor 宋瑾张恒辉孔雅娴
Owner BEIJING IMMUPEUTICS MEDICINE TECH LTD
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