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Methylation marker related to colorectal cancer and kit for detecting colorectal cancer

A methylation marker, colorectal cancer technology, applied in the field of biomedicine, can solve the problems of inconsistent results, limited sensitivity, low concentration, etc.

Active Publication Date: 2021-03-02
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, MethyLight is still susceptible to PCR inhibitors, has limited sensitivity to detect rare methylated alleles against a background of unmethylated alleles, and can be biased during normalization, which can lead to inconsistent results, esp Is in the detection of low levels of DNA methylation (including low concentration and low molecular weight)
Therefore, conventional methylation-specific quantitative PCR often cannot achieve the sensitivity to accurately detect low-level DNA methylation as a cancer biomarker [32]

Method used

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  • Methylation marker related to colorectal cancer and kit for detecting colorectal cancer
  • Methylation marker related to colorectal cancer and kit for detecting colorectal cancer
  • Methylation marker related to colorectal cancer and kit for detecting colorectal cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] Example 1 Screening and verification of cfDNA methylation markers for colorectal cancer

[0125] 1. Colorectal cancer tissue specificity

[0126] The present invention utilizes databases such as TCGA to analyze colorectal cancer tissue-specific methylation profiles, screen out tumor methylation-specific markers, and screen out 10 CpG sites mainly distributed on ZNF304, LIFR and C20orf194 genes. Based on the 450K methylation data of 11 cancer tissues including colorectal cancer in the TCGA database and healthy peripheral blood leukocytes from GEO, it shows that the 10 CpGs are specific to colorectal cancer tissues, and the average methylation level is shown in figure 1 , see Figure 2, Figure 3, and Figure 4 for the box plots of the methylation levels of the sites contained in the three genes. See Table 1 for the genome information of the 10 CpG sites, and Table 2 for the cancer types.

[0127] Table 1

[0128]

[0129]

[0130] Note: Genome annotation information...

Embodiment 2

[0176] Example 2 Methylation-specific fluorescent quantitative PCR (qMSP)

[0177] The qMSP probe method was used to detect the DNA methylation of colorectal cancer, para-cancerous tissues and peripheral blood leukocytes, and primers were designed based on the results of targeted sequencing. The target fragment plasmid was constructed using fully methylated human genomic DNA treated with bisulfite as a template, and the methylation standard curve was constructed by gradient dilution and the equation was established. The sequence without CG sites on ACTB was used as the internal reference gene, and the target gene of the sample was The ratio of the amplified copy number to the amplified copy number of the internal reference gene was used as the relative methylation level of the target gene.

[0178] 1. Primer and probe design

[0179] Design qMSP primers and probes for ZNF304, LIFR and the internal reference gene ACTB for the screened sites, the amplification primers include m...

Embodiment 3

[0209] Example 3 MethyLight ddPCR detection method and establishment of kit

[0210] The technology adopted in the present invention is digital PCR technology combined with the MethyLight method, by designing specific primers and probes for human ZNF304 and LIFR candidate methylated regions, amplifying bisulfite modified and prepared by microdropletization Measure the DNA sample, and determine whether methylation and the methylation level have occurred in the human ZNF304 and LIFR candidate methylation regions in the sample to be tested according to the ratio of the number of positive droplets amplified by PCR and the total number of positive droplets, thereby Determine whether colorectal cancer occurs in the sample to be tested.

[0211] 1. Primer and probe design

[0212] Design ddPCR primers and probes for ZNF304 and LIFR for the screened sites. Amplification primers include methylated sequence amplification primers and unmethylated sequence amplification primers. TaqMan p...

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Abstract

The invention discloses a methylation marker related to colorectal cancer and a kit for detecting the colorectal cancer. The methylation marker is 10 CpG sites distributed on ZNF304 , LIFR and C20orf194 genes. The methylation levels of the C20orf194, ZNF304 and LIFR genes are detected in blood and tissue DNA for the first time, a detection method combining qMSP, digital PCR and MethyLight is usedfor the first time, and the detection method for detecting human ZNF304 and LIFR gene methylation based on qMSP and ddPCR technologies is successfully established by optimizing PCR primers and probesand optimizing a reaction system and reaction conditions. Compared with existing human SEPT 9 gene methylation detection methods based on qPCR technology, the detection method provided by the invention has higher specificity. According to the ddPCR method, qualitative and quantitative detection is carried out on the methylation of the ZNF304 and LIFR genes of a colorectal cancer patient by calculating the number of methylation positive liquid drops and the ratio of the methylation positive liquid drops to the non-methylation positive liquid drops, so that the auxiliary diagnosis of the colorectal cancer is realized.

Description

technical field [0001] The invention relates to a colorectal cancer-related methylation marker and a kit for detecting colorectal cancer, and the invention belongs to the technical field of biomedicine. Background technique [0002] Colorectal cancer is a common malignant disease of the digestive tract. According to GLOBCAN 2018, it is estimated that there will be more than 1.8 million new cases, accounting for about 10% of new cancer cases each year, ranking third in the world, and its mortality rate is second only to lung cancer [1] . It is estimated that there will be more than 140,000 new cases and 50,000 deaths in the United States in 2019, accounting for about one-tenth of all cancer morbidity and deaths [2] . The 5-year survival rate of colorectal cancer can reach more than 65%, and the 5-year survival rate of patients diagnosed and treated early can reach more than 80%, while the 5-year survival rate of patients with stage IV is only 12%. [3] . According to stati...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2600/16C12Q2531/113C12Q2537/143C12Q2545/113C12Q2563/107C12Q2563/159
Inventor 赵亚双李大鹏张磊崔滨滨黄浩王帆
Owner HARBIN MEDICAL UNIVERSITY
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