Lung cancer related methylation gene combination in plasma and application of lung cancer related methylation gene combination

A technology of gene combination and methylation, which is applied in the combination of lung cancer-related methylation genes in plasma and its application fields, can solve the problems of inapplicability and insufficient stability of the method

Pending Publication Date: 2021-01-08
山东康华生物医疗科技股份有限公司
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Other enzyme digestion methods are only used in scientific research, and

Method used

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  • Lung cancer related methylation gene combination in plasma and application of lung cancer related methylation gene combination
  • Lung cancer related methylation gene combination in plasma and application of lung cancer related methylation gene combination
  • Lung cancer related methylation gene combination in plasma and application of lung cancer related methylation gene combination

Examples

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Embodiment 1

[0074] Example 1: Detection of DNA methylation by real-time fluorescent PCR, design and verification of primer probe set

[0075] This embodiment provides a method and process for designing a primer-probe set including six lung cancer-related genes SHOX2, CDO1, PTGER4, TAC1, HOXA7, and SCT and an internal reference GAPDH;

[0076] This example also provides a method and kit for detecting the methylation of lung cancer-related genes in plasma, which can be used for screening and auxiliary diagnosis of lung cancer and early lung cancer. The test results are verified by negative reference products and positive reference products. Verify the primer-probe set designed above.

[0077] Specific steps are as follows:

[0078] 1. Query the 6 lung cancer-related genes and the internal reference GAPDH sequence:

[0079] Lung cancer-related genes SHOX2, CDO1, PTGER4, TAC1, HOXA7, and SCT and the internal reference GAPDH were queried through the National Center for Biotechnology Informat...

Embodiment 2

[0123] Example 2: Construction of target relative methylation standard linear equation

[0124] Preparation of SHOX2 / CDO1 relative methylation degree reference product with GAPDH as reference, mainly including methylation degree of 100% (10 5 copies / ul (SHOX2): 10 5 copies / ul(CDO1): 10 5 copies / ul(GAPDH)), 10% (10 4 copies / ul(SHOX2):10 4 copies / ul (CDO1): 10 5 copies / ul(GAPDH)), 1% (10 3 copies / ul(SHOX2):10 3 copies / ul(CDO1):10 5 copies / ul(GAPDH)), 0.1% (10 2 copies / ul(SHOX2):10 2 copies / ul(CDO1):10 5 copies / ul(GAPDH)), 0.01% (10 1 copies / ul(SHOX2): 10 1 copies / ul(CDO1):10 5 copies / ul(GAPDH)). The relative methylation reference products of the genes of the other four lung cancers are prepared in the same way as SHOX2 / CDO1, so details are not repeated here.

[0125] Using the above-mentioned serially diluted DNA template, carry out qPCR experiment, the reaction system is as follows:

[0126] Table 3 SHOX2 / CDO1 relative methylation detection reaction system

[01...

Embodiment 3

[0131] Example 3: Validation of clinical sensitivity and clinical specificity of 6 different lung cancer methylation gene joint detection kits in detection of lung cancer patients and normal human plasma

[0132] This example judges the clinical sensitivity of the gene combinations selected in this kit by verifying the DNA methylation status of different combinations of 6 lung cancer-related methylation genes in lung cancer plasma and the methylation status of plasma cell-free DNA in normal human plasma and clinical specificity.

[0133] Plasma cell-free DNA extraction:

[0134] use Serum / Plasma Circulating DNA Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., Cat. No. N902-01), operated according to the kit instructions.

[0135] Plasma cell-free DNA bisulfite conversion:

[0136] The sulfite conversion was carried out according to the instruction manual of the EZ DNA Methylation-DirectTM KIT (D5031) of the ZYMO RESEARCH biological company kit. Amplification a...

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Abstract

The invention discloses a lung cancer related methylation gene combination in plasma and application of the lung cancer related methylation gene combination. According to the lung cancer related methylation gene combination in plasma, the combination comprises SHOX2 and CDO1. The combination further comprises any one or more genes selected from the group consisting of PTGER4, TAC1, HOXA7 and SCT.The composition provided by the invention is used as a detection target to be applied to preparation of a lung cancer auxiliary diagnosis kit. The lung cancer auxiliary diagnosis kit is characterizedby comprising the composition provided by the invention. The kit provided by the invention can improve the detection sensitivity of the biomarker to 310 copies/microliter, and the methylation conditions of the six lung cancer related genes SHOX2, CDO1, PTGER4, TAC1, HOXA7 and SCT are meaningful clinical indexes for potential lung cancer (especially early lung cancer) diagnosis, prognosis evaluation and curative effect monitoring.

Description

technical field [0001] The invention belongs to the field of biomedicine detection, and relates to a combination of methylation genes related to lung cancer in blood plasma and an application thereof. Background technique [0002] Lung cancer is a malignant tumor with the highest morbidity and mortality rate in China and even the world, and it is also one of the leading causes of death worldwide, seriously threatening human health and quality of life. [0003] Lung cancer can be roughly divided into two categories: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Lung cancer, large cell lung cancer, accounts for about 80% to 85%, and the rest is small cell lung cancer. All of them are more or less related to abnormal methylation of DNA. [0004] Due to the increasing air pollution caused by the continuous development of my country's industrialization, coupled with the world's highest prevalence of tobacco and the influence of aging and other factors, t...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2600/166C12Q2531/113C12Q2561/101C12Q2523/125C12Q2545/101C12Q2563/107
Inventor 顾月清刘云龙陈红红
Owner 山东康华生物医疗科技股份有限公司
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