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Primer pair, kit and method for colorectal-cancer related gene Septin9 methylation detection

A colorectal cancer, methylation technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome operation, low accuracy, low sensitivity, etc., to save The effect of social resources, sensitive specificity, and rapid detection

Inactive Publication Date: 2018-05-18
韩林志
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the technical problems of cumbersome operation, low accuracy and low sensitivity in the above-mentioned traditional methylation detection method, the present invention provides a simple-to-operate, high-accuracy and high-sensitivity method based on fluorescent PCR technology for colorectal Primer pair, kit and method for detection of cancer-related gene Septin9 methylation

Method used

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  • Primer pair, kit and method for colorectal-cancer related gene Septin9 methylation detection
  • Primer pair, kit and method for colorectal-cancer related gene Septin9 methylation detection
  • Primer pair, kit and method for colorectal-cancer related gene Septin9 methylation detection

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: the preparation of kit

[0036] 1. Design and synthesis of primers and probes

[0037] For the promoter region of the Septin9 gene in the human genome and the internal reference gene β-Actin (b-actin) (for the sequence, refer to the human whole genome sequence published in the NCBI database), use Primer Premier 3.0 and Methyl PrimerExpress v1.0 software to design a pair of Specific primers and probes.

[0038] Specific primers and probe sequences are shown in the table below:

[0039]

[0040]

[0041] Remarks: Y is a merged base, that is, Y=C / T.

[0042] 2. Selection of reference substance

[0043] Using Septin9 methylated standard product DNA as normal human peripheral blood genomic DNA treated with SssI methylase can make C in all CG sequences of the genome methylated at the C5 position; the non-methylated standard product DNA is Genomic DNA from normal human peripheral blood.

[0044] 3. Composition of PCR reaction solution

[0045] Including...

Embodiment 2

[0066] Example 2: Method for detecting colorectal cancer methylation using the above kit

[0067] 1. Technical principle

[0068] A pair of specific primers and probes for methylation detection were designed in the promoter regions of the Septin9 gene and the internal reference gene β-Actin (b-actin) in the human genome. Then use the primers and probes to amplify the sample DNA converted by sulfite, and determine the methylation rate of the sample to be tested according to the relative fluorescence CT value of the PCR amplification results of Septin9 and β-Actin genes. The rate indirectly judges the risk of colorectal cancer.

[0069] 2. Detection method

[0070] Step 1: Take the DNA extracted from the sample to be tested, carry out transformation treatment on it, and use the transformed DNA as a template for PCR;

[0071] Wherein, the reagents used in the conversion treatment are bisulfite or bisulfite, and other auxiliary materials (the corresponding kit is EpiTect Fast D...

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Abstract

The invention relates to a primer pair for colorectal-cancer related gene Septin9 methylation detection. The primer pair comprises a Septin9 forward primer, a Septin9 reverse primer, a Septin9 detection probe, a beta-Actin forward primer, a beta-Actin reverse primer and a beta-Actin detection probe. The invention relates to a kit for colorectal-cancer related gene Septin9 methylation detection. The kit comprises PCR reaction liquid comprising the primers and the probes, and the PCR reaction liquid comprises the forward primers, the reverse primers, the detection probes, 10*PCR buffer, dNTP, nuclease-free water and Ex Taq enzyme. The invention also relates to a method for colorectal-cancer related gene Septin9 methylation detection. The kit and the detection method have the advantages of being accurate in detection result, high in detection flux, good in specificity and sensitivity, short in detection time, convenient to use and capable of effectively meeting the clinical requirements.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnosis, in particular to a primer pair, a kit and a method for detecting methylation of a colorectal cancer-related gene Septin9. Background technique [0002] Septin9 gene is a potential tumor suppressor gene discovered in recent years. It is located on human chromosome 2q23 and is one of the genes that regulate the normal growth of cells. The encoded protein is located in the cytoplasm and is a highly glycosylated protein. It is a downstream signal of p53-mediated cell cycle regulation, which can make abnormal cells stagnate in G2 phase and prevent cell proliferation. Abnormal methylation of Septin9 promoter can inhibit transcription, cause G2 / M checkpoint regulation disorder, and lead to abnormal cell proliferation. Septin9 gene methylation and loss of expression are associated with a variety of cancer or tumor cell lines, including colorectal cancer, gallbladder cancer, lymphoma, colorect...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/154C12Q2600/166C12Q2531/113C12Q2523/125C12Q2563/107C12Q2545/101
Inventor 韩林志肖芳李书
Owner 韩林志
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