Marking composition for detecting colorectal adenoma and early diagnosis reagent thereof

A colorectal and adenoma technology, applied in the field of colorectal advanced adenoma detection kits, can solve the problems of low compliance in colonoscopy screening, cumbersome bowel preparation, high false positive rate, and achieve high diagnostic efficiency , high sensitivity, convenient and simple sampling process

Pending Publication Date: 2022-05-31
SHANGHAI REALBIO TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Colonoscopy is the gold standard for the diagnosis of colorectal cancer, but it is highly invasive and cumbersome to prepare the bowel, and the compliance of colonoscopy screening in China is low
Fecal occult blood (FIT) is currently the most widely used method for colorectal cancer screening, but it has defects such as a high false positive rate.

Method used

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  • Marking composition for detecting colorectal adenoma and early diagnosis reagent thereof
  • Marking composition for detecting colorectal adenoma and early diagnosis reagent thereof
  • Marking composition for detecting colorectal adenoma and early diagnosis reagent thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0053] The inventors determined the target nucleic acid for the diagnosis of colorectal advanced adenoma according to the following methods and steps:

[0054] Step 1, download the 450k methylation chip-based data set of colorectal adenoma from the GEO database, merge the matrix, and calculate the difference between the methylation degree of each methylation site between the adenoma group and the normal group-β value (beta value, from 0 to 1, representing complete demethylation to complete methylation).

[0055] Step 2, methylation region screening. Centering on each CpG site for circular screening, the screened methylation sites need to meet the following conditions:

[0056] 1. Screen for at least three differentially methylated sites within a continuous 20 bp;

[0057] 2. All methylation sites in each region are significantly different;

[0058] 3. All methylated sites in each region were co-enriched in adenoma or normal group.

[0059] Step 3, methylation site screenin...

Embodiment 2

[0065] 1. DNA Extraction from Stool Samples

[0066] Concrete extraction method may comprise the following steps:

[0067] Follow the standard operating procedures of the QIAamp Fast DNA Stool Mini Kit kit:

[0068] Add 1mL InhibitEX Buffer to the sample, vortex for 1min until the sample is fully homogenized, and centrifuge at 16000g for 1min;

[0069] Transfer 25 μL of proteinase K to a new centrifuge tube, pipette 600 μL of the supernatant from the previous step into the proteinase K tube, add 600 μL of Buffer AL, vortex for 15 seconds, and incubate at 70°C for 10 minutes;

[0070] Add 600μL of ethanol, vortex and mix well, divide into QIAampspin column three times, and centrifuge at 16000g for 1min each time;

[0071] Carefully add 500μL Buffer AW1 to the column, centrifuge at 16000g for 1min, and remove the liquid in the collection tube;

[0072] Add 500μL Buffer AW2 to the column, centrifuge at 16000g for 3min, and remove the liquid in the collection tube;

[0073] Co...

Embodiment 3

[0102] The present inventors used the reagents for the early detection of advanced colorectal adenomas, including specific primers and probes for specifically detecting the methylation of the SYNPR gene, MEGF10 gene, LSM2 gene and SLC32A1 gene, and carried out the following method on the sample: Methylation detection, the steps are as follows figure 1 shown, including:

[0103] 1. Sample collection:

[0104] A total of 151 colorectal advanced adenoma samples and 60 healthy controls were collected.

[0105] 2. Sample DNA extraction

[0106] DNA was extracted from the collected samples using a commercial stool sample DNA extraction kit. The specific extraction method is as follows: Follow the standard operating procedure of the QIAamp Fast DNA Stool Mini Kit kit: add 1mL InhibitEX Buffer to the sample, vortex for 1min until the sample is fully homogenized, and centrifuge at 16000g for 1min; take 25μL proteinase K into a new centrifuge tube , and pipette 600 μL of the superna...

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Abstract

The invention belongs to the field of molecular biology, and particularly relates to a tumor marker combination for detecting colorectal progression stage adenoma and a reagent for early diagnosis of colorectal progression stage adenoma. According to the method, excrement is taken as a detection sample, target gene DNA methylation is taken as a molecular marker, the target gene methylation is one or more of SYNPR, MEGF10, LSM2 and SLC32A1 genes, and the methylation level of the target gene methylation is detected. Results show that the provided reagent for detecting single or combined SYNPR, MEGF10, LSM2 and SLC32A1 genes can well distinguish colorectal progression stage adenoma from normal human samples, and can be used for screening and early diagnosis of colorectal progression stage adenoma. The invention also relates to a specific primer and a specific probe for detecting methylation of the SYNPR gene, the MEGF10 gene, the LSM2 gene and the SLC32A1 gene.

Description

technical field [0001] The invention relates to the field of molecular biology technology, in particular to a reagent for detecting advanced colorectal adenoma and its application, and a kit for detecting advanced colorectal adenoma. Background technique [0002] Colorectal cancer (CRC), also known as colorectal cancer, is currently a common malignant tumor. [0003] It is generally believed that the occurrence of CRC needs to go through the development process of normal mucosal hyperplasia-adenoma-adenocarcinoma. The vast majority of colorectal cancers originate from colorectal adenomas. The cure rate of early detection of rectal cancer can reach more than 90%, while that of advanced cancer is about 5%. [0004] Traditional colorectal disease screening mainly includes colonoscopy and fecal occult blood test, all of which have certain limitations. Colonoscopy is the gold standard for the diagnosis of colorectal cancer, but it is highly invasive and cumbersome for intestina...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 吴春燕张笑笑陈煜庶王美丛刘晶
Owner SHANGHAI REALBIO TECH CO LTD
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