DNA methylation marker for early colorectal cancer and adenoma, method for detecting DNA methylation marker and application of DNA methylation marker

A technology for methylation markers and colorectal cancer, applied in recombinant DNA technology, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of high missed detection rate in endoscopic screening and high technical requirements for examiners

Pending Publication Date: 2021-08-20
SHANGHAI GENECHEM CLINICAL LAB INC
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Intestinal adenoma can be screened by endoscopy, but the technical requirements for examiner

Method used

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  • DNA methylation marker for early colorectal cancer and adenoma, method for detecting DNA methylation marker and application of DNA methylation marker
  • DNA methylation marker for early colorectal cancer and adenoma, method for detecting DNA methylation marker and application of DNA methylation marker
  • DNA methylation marker for early colorectal cancer and adenoma, method for detecting DNA methylation marker and application of DNA methylation marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0147] Embodiment 1TaqMan-PCR method detects the methylation site in the stool sample

[0148] 1.1 Extraction and transformation of DNA samples

[0149] Human NDRG4 and ACTIN gene fragments were extracted from fecal samples using magnetic beads linked to specific probes, eliminating the interference of microorganisms and other human DNA. Among them, NDRG4 is used as the detection target, and ACTIN is used as the internal reference.

[0150] The sample used in this example is feces, which is convenient for sampling. The preservation of feces samples uses the prepared sample preservation solution, which can preserve the samples at room temperature for up to 7 days, which reduces the difficulty of sample transportation. Among them, the sequence of the specific probe linked on the magnetic beads is as follows:

[0151]

[0152] Sample Preservation Solution Formula:

[0153] ingredients Sodium acetate Sodium chloride EDTA SDS concentration 0.1M 0.5...

Embodiment 2

[0217] Example 2 MS-HRM method detects methylation sites in tissue samples

[0218] Another method for detecting methylation sites in a sample—methylation-sensitive high-resolution melting curve analysis (MS-High Resolution Melting Curve Analysis) is also provided in this embodiment. The MS-HRM method is a simple and sensitive method for detecting gene methylation levels that does not require subsequent manipulation of PCR products. This method is mainly realized by comparing the melting temperature and peak shape of the curve and can detect a single methylation in a series of CpG sites, and at the same time, it can also detect the methylation of a series of CpG sites level to analyze. The methylation of individual CpG sites and the average methylation level will affect the shape of the melting curve. Before the PCR reaction, design primers at non-CpG island sites, so that the middle of the pair of primers contains meaningful methylated CpG islands. Once these CpG islands ar...

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Abstract

The invention provides a DNA methylation marker for diagnosis, screening and risk prediction of early colorectal cancer and adenoma. The marker is characterized in that a CpG site in an NDRG4 gene is simultaneously methylated on at least two sites of the 38th site, the 47th site, the 50th site and the 52nd site in SEQ ID NO: 1. The invention also provides a method, a primer pair, a probe and a kit for detecting the DNA methylation marker. The method provided by the invention has good specificity and high sensitivity on early colorectal cancer and adenoma, is low in detection cost and simple to operate, and is beneficial to wide application of early colorectal cancer screening.

Description

technical field [0001] The invention relates to the field of gene detection, in particular, the invention relates to a methylation marker of early colorectal cancer and adenoma, a detection method and application thereof. Background technique [0002] In China, the incidence of colorectal cancer ranks fourth among all malignant tumors. Insufficient screening of colorectal cancer in China is the main reason for the low five-year survival rate of colorectal cancer in my country. The latest research shows that feces contain a small amount of nucleic acids derived from human intestinal epidermal exfoliated cells, and multi-target detection of these nucleic acids can effectively predict signs of malignant transformation. For example, multi-target detection of fecal DNA can be used to screen for intestinal cancer , although the sensitivity for high-grade adenomas is low. The fecal DNA multi-target detection technology was approved by the FDA for bowel cancer screening in 2014 an...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/6883C12Q1/686C12Q2600/154C12Q2600/112C12Q2561/101C12Q2563/107
Inventor 何逖吴帅来姚浩
Owner SHANGHAI GENECHEM CLINICAL LAB INC
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