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A detection method to detect methylation of specific genes

A specific gene and detection method technology, applied in the field of detection of specific gene methylation, can solve the problems of high professional requirements for operators, cumbersome operation process, expensive fluorescent PCR equipment, etc., to achieve enhanced sensitivity and simple result analysis , the effect of high sensitivity

Active Publication Date: 2020-07-21
天津康博尔生物基因技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this type of method requires relatively expensive fluorescent PCR equipment, and the operation process is relatively cumbersome. The test cycle of the workflow execution takes about 8 hours, and the professional requirements of the operators are relatively high, so it cannot well meet the actual screening needs.

Method used

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  • A detection method to detect methylation of specific genes
  • A detection method to detect methylation of specific genes
  • A detection method to detect methylation of specific genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of Lateral Flow Nucleic Acid Detection Test Paper

[0030] See attached Figure 1A , wherein, SP: sample pad CP: binding pad NM: nitrocellulose membrane detection area AP: absorbent pad.

[0031] (1) Preparation of nitrocellulose membrane detection area:

[0032] The nitrocellulose detection membrane was cut into long strips, placed on the platform of the dot-spraying apparatus, and digoxinumab, FITC monoclonal antibody and biotin-BSA were sprayed on the detection membrane respectively to form T1, T2 and C lines. Dry at 42°C for 30 minutes or dry naturally at room temperature.

[0033] (2) Preparation of binding pad:

[0034] Cut the glass wool into long strips, place it on the platform of the dot sprayer, take the nanoparticle markers, add a certain volume of pretreatment solution, spray dots on the glass wool, and dry at 50°C for 30 minutes.

[0035] (3) Preparation of sample pad:

[0036] Cut glass wool into long strips, soak with a certain...

Embodiment 2

[0039] Example 2: Methylation-dependent restriction endonuclease treatment of DNA

[0040] (1) Use a commercial DNA extraction kit or other appropriate methods to extract human genomic DNA;

[0041] (2) Take 10 μL DNA solution (100ng-2μg), 5 μL 10x reaction buffer, 2U methylation-dependent restriction endonuclease and 35 μL deionized water, mix well, centrifuge briefly, and place in a PCR instrument for reaction;

[0042] (3) The reaction conditions are as follows:

[0043] The first stage: 30 ℃ 1hr, 1 cycle,;

[0044] The second stage: 70°C for 15 minutes, 1 cycle.

Embodiment 3

[0045] Embodiment 3: Human SEPT9 gene methylation PCR reaction

[0046] (1) Design upstream methylation-specific recognition primers, upstream general amplification primers and downstream gene-specific primers according to the methylation island of the SEPT9 gene promoter as follows:

[0047]

[0048] (2) Prepare a PCR reaction solution according to the following system, mix it and centrifuge briefly, and place it in a PCR instrument for reaction:

[0049]

[0050]

[0051] (3) The reaction conditions are as follows:

[0052] The first stage: 50°C for 5 minutes, 95°C, 1 cycle;

[0053] The second stage: 95°C 15S, 65°C 40S, 40 cycles.

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Abstract

The invention discloses a detecting method for detecting methylation of a specific gene. The detecting method comprises a methylation-specific PCR amplification reaction and side flow nucleic acid detection. The methylation of the specific gene is identified and amplified based on a PCR method; the methylation is detected by combining a side flow technology after an amplification product is marked. The detecting method for detecting the methylation of the specific gene has the beneficial effects that higher detecting sensitivity and specificity than those of similar technologies or products are realized, and the methylation of the specific gene is detected quickly, conveniently, sensitively and economically by adopting a simple and high-efficiency DNA methylation treatment technology and a PCR amplification technology, and utilizing nano-color latex microspheres as a visible signal of nucleic acid detection instead of optical signals, such as a fluorescent probe, without complicated operation and expensive instruments; the detecting method for detecting the methylation of the specific gene is suitable for popularization, application and industrialization.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a detection method for detecting specific gene methylation. Background technique [0002] Malignant tumors (cancers) are one of the diseases that seriously threaten human health. Malignant tumors account for about a quarter of all deaths among Chinese residents, and are on the rise. One of the reasons is that 60%-80% of cancers in China are diagnosed in the middle and advanced stages for the first time, and the five-year survival rate of patients is relatively low. Therefore, screening for cancer at an early stage is of great significance for the diagnosis and treatment of cancer. At present, the conventional screening methods for cancer are mainly tumor marker detection and imaging examination, but these methods are not sensitive enough to cause missed detection, or insufficient specificity to cause misdiagnosis, or cause discomfort to patients or have low cost performance, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/113C12Q2523/125C12Q2565/625C12Q2521/301
Inventor 张亮杨光
Owner 天津康博尔生物基因技术有限公司
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