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Primer pair, kit and method for methylation detection of stomach cancer related genes Reprimo and RNF180

A methylation and kit technology, which is applied to the primer pair for RNF180 methylation detection and the field of gastric cancer-related gene Reprimo, can solve the problems of cumbersome operation, low sensitivity, low accuracy, etc. and the effect of good specificity and improved detection rate

Inactive Publication Date: 2018-04-13
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, high accuracy, high sensitivity and based on fluorescent PCR technology for gastric cancer related Primer pair, kit and method for gene Reprimo, RNF180 methylation detection

Method used

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  • Primer pair, kit and method for methylation detection of stomach cancer related genes Reprimo and RNF180
  • Primer pair, kit and method for methylation detection of stomach cancer related genes Reprimo and RNF180
  • Primer pair, kit and method for methylation detection of stomach cancer related genes Reprimo and RNF180

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1: the preparation of kit

[0038] 1. Design and synthesis of primers and probes

[0039] For the promoter regions of the Reprimo and RNF180 genes in the human genome and the internal reference gene β-Actin (b-actin) (for the sequence, refer to the human genome sequence published in the NCBI database), use Primer Premier 3.0 and MethylPrimer Express v1.0 software to design respectively Specific primers and probes.

[0040] Specific primers and probe sequences are shown in the table below:

[0041]

[0042]

[0043] 2. Selection of reference substance

[0044] Use methylated standard DNA as a positive control substance, wherein the Reprimo and RNF180 methylated standard DNA is normal human peripheral blood genomic DNA treated with SssI methylase, which can make C in all CG sequences of the genome at the C5 position methylated; the non-methylated standard DNA is normal human peripheral blood genomic DNA.

[0045] 3. Composition of PCR reaction solutio...

Embodiment 2

[0069] Example 2: Method for detecting gastric cancer methylation using the above kit

[0070] 1. Technical principle

[0071] A pair of specific primers and probes for methylation detection were designed in the promoter regions of the Reprimo and RNF180 genes and the internal reference gene β-Actin (b-actin) in the human genome. Then use the primers and probes to amplify the sulfite-converted sample DNA, and judge whether the sample is methylated or not by comparing the relative fluorescent CT values ​​of the PCR amplification results of Reprimo and RNF180 with the β-Actin gene.

[0072] 2. Detection method

[0073] 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;

[0074] Among them, the DNA extraction kit is QIAmp DNA Mini Kit (purchased from QIAGEN, Germany); the reagents used for conversion treatment are bisulfite or bisulfite, and other auxiliary (the corresponding ...

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Abstract

The invention relates to a primer pair for methylation detection of stomach cancer related genes Reprimo and RNF180. The primer pair comprises forward primers, reverse primers and detection probes ofReprimo, RNF180 and beta-Actin. The invention relates to the kit for methylation detection of the stomach cancer related genes Reprimo and RNF180. The kit comprises a PCR (polymerase chain reaction) solution of the primers and the probes, and the PCR solution comprises the forward primers, the reverse primers, the detection probes, a 10*PCR buffer, dNTP (deoxy-ribonucleoside triphosphate), Mg<2+>,nuclease-free water and an Ex Taq enzyme. The invention further relates to the method for methylation detection of the stomach cancer related genes Reprimo and RNF180. The kit and the detection method thereof have the advantages of accurate detection result, high sensitivity, high flux, high specificity and short detection time.

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 the methylation of gastric cancer-related genes Reprimo and RNF180. Background technique [0002] Reprimo 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 normal cell growth. 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 Reprimo promoter can inhibit transcription, cause G2 / M checkpoint regulation disorder, and lead to abnormal cell proliferation. Reprimo gene methylation and loss of expression are associated with a variety of cancer or tumor cell lines, including gastric cancer, gallbladder cancer, lymphoma, colorect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/154C12Q2600/166C12Q2545/101C12Q2563/107C12Q2531/113
Inventor 韩林志肖芳许丽媛
Owner 韩林志
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