CN117431315A - Methylation biomarker for colorectal cancer lymph node metastasis detection and detection kit - Google Patents

Abstract

The present invention relates to a DNA methylation biomarker or a combination thereof that can be used for colorectal cancer diagnosis. The DNA methylation biomarker includes any two of ten fragments selected from the CpG-rich region in the GABRA1 gene. or a combination of two or more. The present invention also provides detection kits for the above-mentioned methylation biomarkers or combinations thereof, and detection methods. The DNA methylation biomarkers or combinations thereof of the present invention have good prospects for judging the risk of colorectal cancer by detecting their co-methylation degree, and can reflect the occurrence of colorectal cancer more sensitively and specifically, avoiding the need for imaging. The subjectivity of observation and interpretation results involved in scientific inspections is eliminated, and the accuracy is improved.

Description

Colorectal cancer lymph node metastasis detection methylation biomarkers and detection kits

Technical field

The invention belongs to the field of biotechnology, and specifically relates to methylation biomarkers and detection kits for detecting lymph node metastasis of colorectal cancer.

Background technique

Colorectal cancer, also known as Colorectal Cancer (CRC), is one of the most common malignant tumors in the world, with its morbidity and mortality ranking third and second respectively among all cancers. Colorectal cancer has an insidious onset, with early lesions located in the mucosa and submucosa, and the disease progresses slowly, with obvious clinical symptoms not appearing until the middle and late stages. Its occurrence and development are the result of a comprehensive interaction of multiple factors, multiple genes, and multiple processes, including multiple gene mutations, deletions, and heterozygous losses, as well as epigenetic changes such as gene methylation and histone modifications. The current difficulties in clinical diagnosis and treatment of colorectal cancer mainly lie in the following aspects: ① Colorectal cancer is difficult to detect in time in the early stage; ② Tissue biopsy is invasive, has tumor heterogeneity and is impractical to obtain repeatedly; ③ Conventional tumor markers have sensitivity It has shortcomings such as low and poor specificity; ④ it is impossible to monitor and evaluate the effect of diagnosis and treatment at any time; ⑤ the heterogeneity of tumors limits the guidance of individualized medication and the means of monitoring drug resistance. Recurrence and metastasis are the main reasons for the high mortality rate of colorectal cancer, and about 20% to 25% of patients relapse after surgery.

Colorectal cancer may generally have three types of metastasis: blood type metastasis, lymphatic metastasis and implantation metastasis. Generally, lymphatic metastasis is the main way of tumor metastasis. At present, to determine whether lymph node metastasis occurs in colorectal cancer, colonoscopy can be used to determine whether the scope of colon cancer has infiltrated and spread, and then combined with pelvic imaging examinations, such as enhanced CT or magnetic resonance. These methods are invasive and can easily cause physical discomfort, and patients' compliance with colonoscopy is relatively low. Additionally, the quality of bowel preparation may affect assay sensitivity. Therefore, there is a need to establish a highly accurate diagnostic method for colorectal cancer.

In recent years, due to the continuous advancement of molecular biology technology, great progress has been made in the screening of biomarkers for colorectal cancer detection, including the establishment of a colorectal cancer metastasis prediction system. However, it has not been applied to lymphatic metastasis of colorectal cancer. There are still few biomarkers for patient testing, and even fewer that can be actually used in clinical diagnosis. Screening more biomarkers that can effectively detect lymphatic metastasis of colorectal cancer eliminates the need to obtain additional biological samples, improves patient compliance, and can be used for general screening and early prevention detection in clinical settings.

Contents of the invention

The purpose of the present invention is to provide a set of methylation biomarkers that can be used to determine the occurrence of lymph node metastasis of colorectal cancer and to detect the degree of co-methylation in the DNA of colorectal cancer lymph node metastasis and adjacent tissue. Sensitively and specifically reflects the presence of lymph node metastasis of colorectal cancer.

A first aspect of the present invention is to provide a methylation biomarker that can be used for detecting lymph node metastasis of colorectal cancer. The methylation biomarker is the GABRA1 gene, or is selected from the CpG-rich region of the GABRA1 gene. .

In some embodiments, the CpG-rich region in the GABRA1 gene is at least one selected from the region including the amplification products of the following primer pairs: SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.16 and SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.22 and SEQ ID NO.23, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.28 and SEQ ID NO.29 .

In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9 and 10 of the above regions are included.

In some preferred embodiments, the methylation biomarkers SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.13 and SEQ ID NO. 14. One of SEQ ID NO.16 and SEQ IDNO.17. Further preferred is a combination of the corresponding regions of SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.8, and more preferred is a combination of the above four regions.

A second aspect of the present invention provides the use of the above-mentioned methylation biomarkers and/or their reagents related to detecting methylation levels in preparing a kit for detecting lymph node metastasis of colorectal cancer.

A third aspect of the present invention provides a kit for detecting lymph node metastasis of colorectal cancer.

A kit for colorectal cancer lymph node metastasis, the kit includes a reagent for detecting the methylation level of the above-mentioned DNA methylation biomarker.

In some embodiments, the kit includes the method using ordinary PCR amplification method, fluorescent quantitative PCR method, digital PCR method, DNA methylation chip, liquid phase chip method, sequencing method, methylation chip method or their combinations. Combine the reagents used.

In some embodiments, the sequencing methods include first-generation sequencing, third-generation sequencing, second-generation sequencing, pyrosequencing, bisulfite conversion sequencing, simplified bisulfite sequencing, targeted DNA methyl chemical sequencing.

In some of these embodiments, fluorescence quantitative PCR is used, and the primer pairs and probes are as follows:

SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3;

SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.6;

SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9;

SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO.12;

SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15;

SEQ ID NO.16 and SEQ ID NO.17, SEQ ID NO.18;

SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21;

SEQ ID NO.22 and SEQ ID NO.23, SEQ ID NO.24;

SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27;

SEQ ID NO.28 and SEQ ID NO.29, SEQ ID NO.30.

The present invention provides ten fragments selected from the CpG-rich region occurring in the GABRA1 gene in colorectal cancer lymph node metastasis as specific methylation biomarkers for detection, and designs the above-mentioned methylation biomarkers. Specific primers and probes for methylation detection. The degree of co-methylation of the methylated regions marked by these markers is correlated with lymph node metastasis of colorectal cancer, and can be used to detect or monitor lymph node metastasis of colorectal cancer or predict prognosis or drug efficacy, and has good application prospects. .

Description of the drawings

Figure 1: 10 DNA methylation biomarkers for CpG-rich regions in the GABRA1 gene.

Figure 2: Differences in co-methylation of multiple DNA methylation regions between cancers and adjacent tissues at risk of colorectal cancer recurrence and cancer and adjacent tissues without risk of colorectal cancer recurrence.

Detailed ways

In order to facilitate an understanding of the invention, the invention will be described more fully below. The invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough understanding of the present disclosure will be provided.

The experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing method Conditions recommended by the manufacturer. Various commonly used chemical reagents used in the examples are all commercially available products.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings commonly understood by those skilled in the technical field belonging to the present invention. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In the present invention, "methylation" refers to methylation of cytosine at C5 or N4 of cytosine, methylation of N6 of adenine or other types of nucleic acid methylation. In vitro amplified DNA is usually unmethylated because generally in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA that is unmethylated or methylated from the original template, respectively.

Thus, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, where the methyl moiety is not present on the recognized typical nucleotide bases middle. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide and 5-methylcytosine is.

Methylation status may optionally be represented or indicated by a "methylation value" (eg, representing methylation frequency, fraction, ratio, percentage, etc.). Methylation values can be quantified, e.g., after restriction digestion with methylation-dependent restriction enzymes, or by comparing amplification profiles after bisulfite reaction, or by comparing bisulfite treatment and Unprocessed nucleic acid sequences are generated. Therefore, values such as methylation values represent methylation status and can therefore be used as quantitative indicators of methylation status in multiple copies of a locus. The degree of co-methylation is expressed or indicated by the methylation status of more than one methylation site. Within a methylation region, when the methylation status of more than one methylation site is methylated is defined as comethylation.

As used herein, the term "bisulfite reagent" refers to a reagent that in some embodiments includes bisulfite, disulfite, hydrogen sulfite, or a combination thereof , after DNA is treated with bisulfite reagent, its unmethylated cytosine nucleotides will be converted into uracil, while the methylated cytosine and other bases will remain unchanged, so it can distinguish CpG nucleotides such as Methylated and unmethylated cytidines in nucleotide sequences.

The term "methylation assay" or "methylation level detection" refers to any assay used to determine the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

In embodiments of the present invention, a new set of specific methylation biomarkers related to the occurrence of lymph node metastasis of colorectal cancer is provided.

In some embodiments, the DNA methylation biomarker is derived from the GABRA1 gene.

In some embodiments, on the basis that the DNA methylation biomarker is derived from the GABRA1 gene, the DNA methylation biomarker is selected from a CpG-rich region.

In some embodiments, on the basis that the DNA methylation biomarker is selected from a CpG-rich region in the GABRA1 gene, the DNA methylation biomarker includes any two of the ten fragments in the selected region. one or a combination of two or more.

On the basis of the above, in some of the embodiments, the ten fragments were subjected to a series of DNA methylation analyzes through bisulfite cloning and sequencing, as well as SYBR Green-based MSP assay using several different types of samples. definite.

On the basis of the above, in some embodiments, the different types of samples are colorectal cancer tissue and surrounding normal tissue, advanced adenoma, benign polyps and buffy coat samples.

Some implementations of the present invention involve the use of the above-mentioned DNA methylation biomarkers or combinations thereof and/or detection-related reagents in preparing kits for detecting colorectal cancer.

In some implementations of the present invention, it relates to a kit for detecting the occurrence of colorectal cancer, which kit includes a reagent for detecting the methylation level of the above-mentioned DNA methylation biomarker or a combination thereof.

In some embodiments, the reagents include primers and probes for fluorescence quantitative PCR detection of DNA methylation biomarkers.

The method for detecting methylation biomarkers includes the following steps:

(1) Extract genomic DNA from the sample to be tested;

(2) Treat the extracted genomic DNA with bisulfite to obtain transformed DNA;

(3) PCR amplify the transformed DNA using an amplification primer for the DNA methylation biomarker to obtain a PCR amplification product;

(4) Using the multiplex PCR amplification product as a template, perform fluorescence quantitative PCR amplification with amplification primers and probes targeting the DNA methylation biomarker, collect fluorescence signals, and analyze them.

A method for detecting or diagnosing or predicting colorectal cancer, comprising the following steps,

Extract genomic DNA from the biological sample to be tested;

subjecting the DNA to bisulfite conversion;

The bisulfite-converted DNA is subjected to co-methylation detection using the DNA methylation marker combination to obtain a methylation map;

The methylation profile of the methylation marker combination is compared with the profile determination threshold obtained from mathematical modeling based on the data set to determine the presence of colorectal cancer in the biological sample.

Among them, the co-methylation detection methods include: MSP (methylation-specific PCR), DNA methylation chip, targeted DNA methylation sequencing, digital PCR quantitative and fluorescence quantitative PCR.

The subject sample is blood, plasma, saliva, serum, urine or tissue. Preferably, the tissue includes colorectal cancer tissue and/or colorectal para-cancer tissue.

The present invention also provides a detection kit for detecting the methylation degree of a target methylated region. In the kit, the primer pair and probe have good amplification effect, and the corresponding amplified fragment has good specificity related to the occurrence of colorectal cancer lymph node metastasis as a marker.

The present application will be further elaborated below through specific examples, but shall not be used to limit the protection scope of the present invention.

Example 1

A co-methylation test kit for biomarkers used in colorectal cancer lymph node metastasis detection, including specific primer pairs and probes for co-methylation of multiple methylation regions, as shown in Table 1.1:

Table 1.1 Primer and probe sequences used for mqMSP assay

Internal reference primers and probes are shown in Table 1.2:

Table 1.2 Internal reference primers and probes

The primer probe of the present invention was synthesized by Sangon Bioengineering Co., Ltd. Multiplex PCR reaction reagents were purchased from NEB Company.

Example 2 Fluorescence quantitative PCR for methylation detection

Use commercial fully methylated (positive control) and non-methylated (negative control) standards (purchased from QIAGEN) to detect co-methylation of every 2-3 methylated regions in the methylated region. .

The specific process is as follows:

1. DNA extraction

The extraction kit was purchased from QIAGEN Company and was performed according to the instructions of the kit. The extracted DNA samples were stored in a low-temperature refrigerator at -20°C.

2. DNA bisulfite conversion

The DNA bisulfite conversion kit was purchased from Zymo Company and was carried out according to the instructions of the kit to obtain the transformed DNA.

3. Multiplex PCR amplification

1) Use the primer pair of the methylated region in Table 1 to perform multiplex PCR in one reaction well to amplify the target sequence containing the target region.

2) Configure a PCR primer mixture with a single primer concentration of 5 μM (each primer), which contains forward and reverse primers for each methylated region in the multiplex reaction, totaling 1 reaction well.

3) PCR mixture configuration: Prepare the PCR mixture according to Table 2.1. Do not add DNA to it.

Table 2.1 PCR mixture configuration scheme

Reagents Final concentration Volume(μL) DEPC water / 18.0 5x PCR buffer 1X 10.0 25mM MgCl ₂ 0.25mM 0.5 25mM dNTP mixture 250μM 0.5 5μM forward primer 0.5μM 2.5 5μM reverse primer 0.5μM 2.5 5U/μl PCR enzyme 5Unit 1.0 volume / 35.0

3) Add the transformed DNA sample: Add 35 μL of PCR mixture to the PCR reaction well, and add the transformed DNA to it. The loading volume before DNA conversion is 15 ng, and the total PCR reaction volume is 50 μL. Vortex and centrifuge.

4) PCR reaction program: 96°C for 30 seconds; 96°C for 15 seconds, 65°C for 15 seconds, 72°C for 15 seconds, 30 cycles; 72°C for 5 minutes; store at 4°C for later use.

4. Fluorescent quantitative PCR assay

1) Primers and probes for 10 methylated regions (see Table 1.1 for sequences), and primers and probes for internal control. Each methylated region should be prepared at a final concentration of 10 μM for each primer and 5 μM for each probe. Concentrations are formulated into a set mix.

2) The qPCR reaction solution configuration is as follows:

Table 2.2 PCR mixture configuration scheme

Reagents Volume (μL) DEPC water 2 2X PCR Master Mix 5.00 upstream primers and downstream primers 0.5 probe 0.5 volume 8.00

3) Add DNA sample: Add 8 μL of PCR mixture to the PCR reaction well, and add 2 μL of twice-diluted multiplex PCR product to it. The total volume of PCR reaction is 10 μL. Vortex and centrifuge.

4) Fluorescence quantitative PCR reaction program: 95°C for 5 minutes; 95°C for 20 seconds, 62°C for 60 seconds, and fluorescence signal collection at 62°C, 30 cycles.

Each sample was tested in duplicate 4 times to determine the sensitivity of the biomarker. At the same time, the 0% methylation ratio sample was used to detect the amplification specificity of the sample. The results showed that the negative control was not detected in all combinations and individual quantifications, and the positive control results were completely accurate.

Example 3 used tissue-embedded sections to verify co-methylation detection of 10 methylated regions in patients with colorectal cancer lymphoma metastasis.

1: Sample source:

A total of 30 paraffin-embedded tissue samples of cancer tissue from clinically diagnosed patients with lymphoma metastasis of colorectal cancer were collected; a total of 25 samples of colorectal cancer and adjacent cancer samples from people without lymph node metastasis were collected. All samples had no history of other primary organ tumors.

2. Experimental method:

The detection method described in Example 2 was used to conduct co-methylation detection of 10 methylated regions on the transformed DNA of the above 55 tissues. Among them, the C _T value of each methylated region detected is corrected by the internal reference C _T value, and the relative cycle number of the target region dC _T =C _T (target region)-C _T (internal reference) is obtained; if the target region is not detected , then the relative cycle number dC _T =35 is assigned to the target area.

3: Result analysis:

Figure 2 shows the differential distribution of co-methylation levels of 10 methylated regions in 55 tissue DNA samples detected between people without risk of colorectal cancer recurrence and those with risk of colorectal cancer recurrence. The results in Figure 2 show that there are significant differences in 10 methylation regions between people at risk of colorectal cancer recurrence without lymph node metastasis and those at risk of colorectal cancer recurrence.

In addition, using a single biomarker, the above 30 cases of colorectal cancer lymphoma metastasis paraffin-embedded tissue samples were detected using the primers and probes of the present invention, and data analysis was performed.

The predictive performance AUC of the 10 methylated regions for lymph node metastasis of colorectal cancer in the above clinical tissue samples reached 0.65, with an average value of around 0.71. Among them, areas 2, 3, 5, and 6 are 0.83, 0.75, 0.73, and 0.76 respectively, and have the best detection results; areas 1, 4, and 7-10 are between 0.65 and 0.70, respectively, and are 0.69, 0.65, 0.67, and 0.68. , 0.65, 0.68, its effect is slightly worse than areas 2, 3, 5, and 6. The specific analysis is as follows.

When region 2 was used as a separate biomarker for colorectal cancer lymph node metastasis detection, the AUC was 0.83. When the specificity is 90%, the sensitivity is 75.8%; when the specificity is 95%, the sensitivity is 63.6%.

When region 3 was used as a single biomarker for colorectal cancer lymph node metastasis detection, the AUC was 0.75. When the specificity is 90%, the sensitivity can reach 79.3%; when the specificity is 95%, the sensitivity is also 70.7%.

When region 5 was used as a separate biomarker for colorectal cancer lymph node metastasis detection, the AUC was 0.73. When the specificity is 90%, the sensitivity can reach 72.4%; when the specificity is 95%, the sensitivity is 70.7%.

When region 6 was used as a single biomarker for colorectal cancer lymph node metastasis detection, the AUC was 0.76. When the specificity is 90%, the sensitivity can reach 78%; when the specificity is 95%, the sensitivity is 72.8%.

The above results indicate that the methylation levels of these four biomarkers are highly correlated with lymph node metastasis of colorectal cancer. The other 6, although not as good as these 4, can also be related to lymph node metastasis of colorectal cancer and can be used to detect lymph node metastasis of colorectal cancer individually or in combination.

Furthermore, based on the above results, mathematical modeling of methylation region combinations was performed on the co-methylation relative cycle number d-CT values of the four methylated regions with better detection results and the other six methylated regions. Analysis to find better combinations of methylated regions for the detection of lymph node metastasis in colorectal cancer. Specifically, the AUC value and the determination threshold for dividing the region are calculated based on the ROC curve of a single methylation region. The sensitivity, specificity and Youden index of the methylated region were calculated based on the threshold comparison standard diagnosis. At the same time, 2-60 methylation biomarkers are selected based on the relative cycle number dC _T value of the co-methylation of these 10 methylation regions for exhaustive threshold combination or logistic regression or random forest model fitting, and the fitting equation It can be used to calculate a lymph node metastasis risk score for each sample and is used to detect the occurrence of lymph node metastasis in colorectal cancer.

According to the test results in Table 3.1, it can be seen that the combination of multiple regions as biomarkers has better diagnostic performance for colorectal cancer lymph node metastasis, that is, it has better sensitivity or specificity, especially when regions 2+3+ When 5+6 are combined, the sensitivity and specificity are already very good, and when 10 methylation biomarkers are combined together, the detection effect is the best.

Table 3.1 Combination of multiple methylation regions for detection of lymph node metastasis of colorectal cancer

In addition, the detection method described in this example can be used for parallel detection of 2-10 methylated regions according to the combination scheme of Example 2. The detection method is flexible, simple and easy to combine and match methylated regions.

The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.

Claims (10)

1. A methylation biomarker for colorectal cancer lymph node metastasis detection, characterized in that the methylation biomarker is a GABRA1 gene or is a CpG-rich region selected from the GABRA1 gene.

2. The methylation biomarker of claim 1, wherein the CpG-rich region of the GABRA1 gene is at least one selected from the group consisting of regions of amplification products comprising the following primer pair: SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.16 and SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.22 and SEQ ID NO.23, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.28 and SEQ ID NO.29, preferably one of SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.16 and SEQ ID NO.17 or a combination thereof.

3. The methylation biomarker of claim 2, wherein the CpG-rich region of the GABRA1 gene is a region comprising the amplification product of the following primer pair: SEQ ID No.1 and SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5, SEQ ID No.7 and SEQ ID No.8, SEQ ID No.10 and SEQ ID No.11, SEQ ID No.13 and SEQ ID No.14, SEQ ID No.16 and SEQ ID No.17, SEQ ID No.19 and SEQ ID No.20, SEQ ID No.22 and SEQ ID No.23, SEQ ID No.25 and SEQ ID No.26, SEQ ID No.28 and SEQ ID No.29.

4. Use of a methylation biomarker according to any of claims 1 to 3 and/or a reagent thereof for detecting methylation level in the preparation of a kit for detecting colorectal cancer lymph node metastasis.

5. A kit for colorectal cancer lymph node metastasis, characterized in that the kit comprises a reagent for detecting the methylation level of the methylation biomarker according to any of claims 1 to 3.

6. The kit according to claim 5, wherein the kit comprises reagents used by a general PCR amplification method, a fluorescent quantitative PCR method, a digital PCR method, a DNA methylation chip method, a liquid chip method, a sequencing method, a methylation chip method, or a combination thereof.

7. The kit of claim 6, wherein the sequencing method comprises a generation sequencing method, a second generation sequencing method, a pyrosequencing method, a bisulfite conversion sequencing method, a reduced bisulfite sequencing method, a targeted DNA methylation sequencing method.

8. The kit of claim 5, wherein the reagents comprise primers and probes for fluorescent quantitative PCR detection of a methylation biomarker, the primer pair and probes being selected from at least one of the group consisting of:

SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3;

SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.6;

SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9;

SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO.12;

SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15;

SEQ ID No.16 and SEQ ID No.17, SEQ ID No.18;

SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21;

SEQ ID NO.22 and SEQ ID NO.23, SEQ ID NO.24;

SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27;

SEQ ID No.28 and SEQ ID No.29, SEQ ID No.30.

9. The kit for diagnosis and detection of colorectal cancer according to claim 8, further comprising a primer and a probe for fluorescent quantitative PCR detection of a reference gene.

10. A method of detecting a methylated biomarker according to any of claims 1 to 3, comprising the steps of:

(1) Extracting genome DNA from a sample to be detected;

(2) Bisulphite treatment is carried out on the extracted genome DNA to obtain converted DNA;

(3) Performing PCR amplification on the converted DNA by using an amplification primer aiming at the DNA methylation biomarker of any one of claims 1 to 3 to obtain a PCR amplification product;

(4) Fluorescent signals are collected using PCR amplification products as templates for fluorescent quantitative PCR amplification with amplification primers and probes for the methylation biomarkers of any of claims 1-3.