Nucleic acid combination and kit for colorectal cancer gene methylation detection

A colorectal cancer, methylation technology, applied in recombinant DNA technology, biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of poor sensitivity, low specificity, and single detection sensitivity of adenomas To achieve the effect of improving specificity, high detection specificity, and good comprehensive detection effect

Active Publication Date: 2022-01-28
JIANGSU COWIN BIOTECH CO LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing colorectal cancer-related methylation detection technology has the problem of single marker or low detection sensitivity or specificity, resulting in poor sensitivity in early colorectal cancer or advanced adenoma, which cannot meet the needs of early diagnosis of colorectal cancer, The need for early treatment

Method used

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  • Nucleic acid combination and kit for colorectal cancer gene methylation detection
  • Nucleic acid combination and kit for colorectal cancer gene methylation detection
  • Nucleic acid combination and kit for colorectal cancer gene methylation detection

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1. Collection and storage of feces, DNA extraction from feces

[0050] 1. Feces collection and storage

[0051] Stool sample collection steps are as follows:

[0052] (1) Defecation: Pass the feces into a squatting pan or waste box, and then take a sample immediately;

[0053] (2) Digging feces: Open the fecal DNA sample preservation tube, and use the sampling spoon on the lid to dig out a spoonful of feces (about the size of a jujube);

[0054] (3) Tighten the tube cap: Put the spoon and the stool sample back into the tube, tighten the tube cap, shake and mix for about 30 seconds.

[0055] The fecal DNA sample preservation tube used in this step can use conventional preservation tubes in the field, but it is recommended to use the feces sample collection tube of Jiangsu Kangwei Century Biotechnology Co., Ltd., the product number is CWY041. After using this product to collect feces, you can Store at 2-37°C for at least 60 days, and store at -20°C and -80°C fo...

Embodiment 2

[0070] Example 2. DNA methylation conversion

[0071] The DNA methylation conversion of stool samples can use conventional commercial kits in the field; it is recommended to use the DNA methylation conversion kit of Jiangsu Kangwei Century Biotechnology Co., Ltd., the product number is CWY105. The DNA methylation conversion steps of stool samples are as follows:

[0072] (1) Equilibrate the DNA sample that needs to be methylated to room temperature in advance; take 20 μL of the sample and add it to a new PCR tube;

[0073] (2) Add 180 μL of transformation solution to the sample, with a total volume of 200 μL, mix well and put it into the PCR instrument to start the transformation; the transformation procedure is as follows: 98°C for 10 minutes; 54°C for 1 hour; 4°C hold;

[0074] (3) Take a new centrifuge tube, add 600 μL of buffer MB, transfer the transformation liquid obtained from the reaction in step (2) into buffer MB, shake and mix;

[0075] (4) Add 20 μL of magnetic b...

Embodiment 3

[0081]Example 3. Primer, probe design and performance evaluation for methylated gene detection

[0082] The methylation of genes mainly occurs in the promoter region, and the gene promoter region often contains multiple CpG islands. There is no clear theory that choosing which CpG island as the detection region has better diagnostic performance. In this study, by analyzing the promoter regions of three genes, two different methylated regions were obtained for each gene, named a and b regions.

[0083] 1. Design specific primer pairs and probes

[0084] Colorectal cancer-specific methylation markers septin9, SDC2, and NDRG4 were obtained for detection by reviewing literature, referring to relevant domestic and foreign methylation detection products, and searching the TCGA (The cancer genomeatlas) database for bioinformatics mining markers, etc. For markers, the promoter regions of three markers were obtained through NCBI, and after obtaining two CpG islands a and b for each ge...

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Abstract

The invention provides a nucleic acid combination and a kit for colorectal cancer gene methylation detection, and relates to the technical field of gene detection. The nucleic acid combination comprises a first specific primer pair and a first specific probe which are used for detecting Septin9 gene methylation, a second specific primer pair and a second specific probe which are used for detecting SDC2 gene methylation, and a third specific primer pair and a third specific probe which are used for detecting NDRG4 gene methylation, wherein the base sequence of the first specific primer pair, the base sequence of the second specific primer pair, the base sequence of the third specific primer pair, the base sequence of the first specific probe, the base sequence of the second specific probe and the base sequence of the third specific probe are modified through locked nucleotides. According to the invention, methylation sites in the primer and the probe are subjected to locked nucleic acid modification, so that the detection specificity is greatly improved; and three genes are subjected to joint detection, so that the best detection effect is achieved.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a nucleic acid combination and a kit for detection of colorectal cancer gene methylation. Background technique [0002] Colorectal cancer is currently the third most common cancer in the world. According to the latest national cancer statistics in 2020 from the National Cancer Center, the top ten cancers with the number of new cancer cases in China in 2020 are: lung cancer 820,000, colorectal cancer 560,000 , 480,000 gastric cancers, 420,000 breast cancers, 410,000 liver cancers, 320,000 esophageal cancers, 220,000 thyroid cancers, 120,000 pancreatic cancers, 120,000 prostate cancers, and 110,000 cervical cancers. These ten cancers account for 78% of new cancers %. [0003] The occurrence and development of colorectal cancer mostly follow the "adenomas-carcinoma" sequence, and it generally takes 5 to 10 years for the progression from precancerous lesions to cancer, which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/154C12Q2600/166
Inventor 秦宏亮马竣王春香
Owner JIANGSU COWIN BIOTECH CO LTD
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