WO2021004215A1 - Multi-gene combined detection reagent - Google Patents

Abstract

Disclosed by the present application are a methylation detection reagent, kit and an application thereof. In the present application, by detecting the methylation level in a gene combination of SDC2, COL4A1/COL4A2 and ITGA4, colorectal cancer specimens may be very well distinguished from among stool samples. This application uses a methylation detection reagent comprising the gene combination to detect colorectal cancer.

Description

A multi-gene combined detection reagent Technical field

This application belongs to the field of biomedicine, and particularly relates to primers, capture reagents, nucleic acid probes, methylation detection reagents, kits and applications thereof.

Background of the invention

Colorectal cancer, also known as colorectal cancer, is a common malignant tumor of the digestive tract. The incidence rate is increasing year by year in my country. In some coastal areas of my country, such as Shanghai and Guangzhou, the incidence of colorectal cancer has jumped to the second place, second only to lung cancer. It is currently believed that the formation of bowel cancer is the result of accumulation of genetic defects and epigenetic defects. Early onset of colorectal cancer is hidden, often without obvious symptoms, and symptoms such as blood in the stool, abdominal pain, and diarrhea may appear in the late stage. When the symptoms appear, it is often in the late stage, which brings great pain and expensive treatment to the patient. Therefore, early detection, early diagnosis and early treatment are important measures to reduce the incidence and mortality of colorectal cancer.

Screening can detect bowel cancer and precancerous lesions early and remove the lesions, thereby preventing the occurrence of bowel cancer. The current screening methods for colorectal cancer mainly include occult blood test and colonoscopy. The occult blood test is vulnerable to food or the detection rate of adenomas is not high. Although colonoscopy is the gold standard for the diagnosis of bowel cancer, it is not highly adherent to the population when used as a screening method. Therefore, there is an urgent need for a colorectal cancer screening method with high accuracy and high compliance.

Fecal genetic testing, as a new screening method for bowel cancer, is now receiving more and more attention. this method

It was included in the U.S. bowel cancer screening guidelines in 2016. This method is convenient, non-invasive, and has the characteristics of high detection rate for colon cancer and precancerous adenomas. To make a high-performance stool gene detection kit for colorectal cancer detection, two major obstacles need to be overcome: stool DNA extraction and marker selection. On the one hand, the composition of feces is complex, there are many inhibitors to downstream reactions, and there are many bacterial DNA. To extract human target genes from such a mixture, a set of highly sensitive gene extraction and purification methods is required; on the other hand, There are many markers related to colorectal cancer, especially DNA methylation markers, because studies have shown that DNA methylation is an early event of tumor formation. However, many methylation markers perform well at the cell and tissue level. When used in screening media such as feces and blood, their sensitivity and specificity for colorectal cancer are reduced. For example, the vimentin gene, which is The sensitivity in fecal specimens is 83%, which drops to 46% in stool specimens (J Natl Cancer Inst. 2005 Aug 3; 97(15): 1124-32.).

In addition, because the pathogenesis of colorectal cancer is more complicated, it is a polygenic genetic disease. The existing methods for detecting single methylation markers of colorectal cancer have the following two limitations: First, the existing research and product detection methods are to detect a marker in a PCR reaction well, and the detection method cannot Realize multiple detection of methylation markers in a PCR reaction well. Second, when the existing methylation markers for colorectal cancer detection, such as SDC2, ITAG4, Septin 9, etc., detect colorectal cancer by detecting the methylation level of a single gene, the sensitivity of the detection is limited by genetic factors. The detection sensitivity of methylated SDC2 gene is 84.2%, and the specificity is 97.9%; the detection sensitivity of methylated ITGA4 is 83.8%, and the specificity is 95.2%; the detection sensitivity of methylated Septin9 is 79.3%. 94.3%. The specificity of these single markers for colorectal cancer detection can reach more than 90%, but when the specificity reaches more than 90%, the sensitivity cannot reach more than 90%.

Although a single methylation marker of colorectal cancer can detect part of colorectal cancer, it is necessary to combine colorectal cancer-related markers to detect as many colorectal cancer patients through molecular biology methods, but what you need to face The problem is: 1. The detection value of most colorectal cancer gene methylation markers for colorectal cancer shows overlap in performance, that is, the detection status of the same colorectal cancer patient sample is consistent in most methylation markers of. Therefore, it is very difficult to screen gene methylation markers that can complement each other in colorectal cancer detection capabilities, and a lot of research work is required. In addition, the more gene methylation markers, the false positive phenomena of the detection system will also be superimposed, and the specificity of the detection will be reduced. Therefore, it is very difficult to improve the sensitivity of colorectal cancer detection under the premise of certain detection specificity. 2. It is necessary to balance detection cost, sensitivity and specificity.

Chinese patent application CN109207592A analyzed multiple markers to detect colorectal cancer models. Among them, the sensitivity of the combined detection of SEPT9, NDRG4, and SDC2 is only 88.6%, and the specificity is only 89.1%; KRAS, BMP3, NDRG4, SEPT9, and SDC2 Among many different combination detection models, the highest AUC value of the ROC curve is only 0.912, and to obtain this 0.912 AUC requires simultaneous joint detection of 5 genetic markers.

Therefore, selecting a combination of markers with extremely high sensitivity and specificity for colorectal cancer detection in stool is the key to the detection of colorectal cancer stool genes, and such a combination of markers is expected to be truly used in the clinical detection of colorectal cancer, and is good The design of primers, capture sequences and probes is also one of the influencing factors that affect the function of the marker combination.

Summary of the invention

In one aspect, this application provides a primer and its application in preparing reagents or kits for detecting colorectal tumors.

In one aspect, this application provides a capture sequence and its application in preparing reagents or kits for detecting colorectal tumors.

In one aspect, the present application provides a probe and its application in preparing reagents or kits for detecting colorectal tumors.

In one aspect, this application provides a multi-gene methylation combined detection reagent, the genes are SDC2, COL4A1/COL4A2 and ITGA4.

On the one hand, this application provides a colorectal tumor detection reagent and kit with strong specificity and high sensitivity.

In some embodiments, the present application provides a primer, which includes a primer such as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and their complementary sequences have at least 85% or at least 90% or at least 91% or at least At least any one of 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99% or 100% identical sequence.

In some embodiments, the primers include SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25 have at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least Any one of a sequence that is 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identical.

In some embodiments, the primers include SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 18 and SEQ ID NO: 19 and SEQ ID NO: 24 and SEQ ID NO: 25 have at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least At least one primer pair in a sequence that is 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identical.

In some embodiments, the primers include SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 11 and SEQ ID NO: 12. .

In some embodiments, the primers include the primer pairs shown in SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 19, and SEQ ID NO: 24 and SEQ ID NO: 25 .

In some embodiments, this application also provides a capture sequence, the capture sequence includes the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, And their complementary sequence has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or At least any one of the 100% identical sequences.

In some embodiments, the capture sequence includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, which has at least 85% or at least 90% or at least 91% or At least one of at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence.

In some embodiments, the capture sequence includes the sequence shown in SEQ ID NO: 1, SEQ ID NO: 6 and SEQ ID NO: 10.

In some embodiments, the capture sequence includes the sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10.

In some embodiments, the present application also provides a nucleic acid probe, the nucleic acid probe comprising the sequence shown in SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, and their complements The sequence has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identity At least any one of the sequence.

In some embodiments, the nucleic acid probe includes at least one of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13.

In some embodiments, the nucleic acid probe comprises the sequence shown in SEQ ID NO: 5, SEQ ID NO: 9 and SEQ ID NO: 13.

In some embodiments, the application also provides the application of the above-mentioned primers or capture sequences or probes in preparing reagents or kits for detecting colorectal tumors.

In some embodiments, the application also provides applications of the above-mentioned primers or capture sequences or probes or reagents or kits.

In some embodiments, the application also provides the application of the above-mentioned primers or capture sequences or probes or reagents or kits in the detection of colorectal tumors.

In some embodiments, this application also provides a multi-gene methylation combined detection reagent, including a combination of SDC2, COL4A1/COL4A2 and ITGA4 gene combination detection reagent.

In some embodiments, the obtained capture sequences, primers and/or probes for each gene in the combination of SDC2, COL4A1/COL4A2 and ITGA4 genes are included.

In some embodiments, capture sequences, primers, and/or probes obtained for the CpG islands of each gene in the SDC2, COL4A1/COL4A2, and ITGA4 gene combination are included.

In some embodiments, the methylation detection reagents provided in the present application detect the morphology of each gene in the combination of SDC2, COL4A1/COL4A2, and ITGA4 genes, intergenic regions or promoter regions, and regions near the promoter regions. Basic level.

In some embodiments, the methylation detection reagent provided in the present application includes a capture sequence obtained from the promoter region of each gene in the SDC2, COL4A1/COL4A2, and ITGA4 gene combination or the CpG island in the vicinity of the promoter region , Primers and/or probes.

In some embodiments, SDC2, COL4A1/COL4A2, and ITGA4 can each be detected for methylation levels. In other embodiments, SDC2, COL4A1/COL4A2, and ITGA4 can be simultaneously detected for methylation levels, such as using multiple real-time fluorescent quantitative PCR to detect the methylation levels of multiple genes. In some aspects, it is more convenient for SDC2, COL4A1/COL4A2 and ITGA4 to be detected simultaneously by multiplex PCR.

"COL4A1/COL4A2" in this application means "COL4A1 or COL4A2". The human genes COL4A1 and COL4A2 are closely linked at the far end of the long arm of chromosome 13. These two genes are in a "head-to-head" positional relationship, and they are transcribed in opposite directions. The 5'ends of the COL4A1 and COL4A2 genes are close to each other, with an interval of 127bp. This sequence is a bidirectional promoter region shared by the two genes [1]. Therefore, the sequence designed according to the bidirectional promoter shared by the two can target both COL4A1 and COL4A2. In this application, whether COL4A1 or COL4A2 is used to label sequence information, there is no difference between the two.

"Detection" in this application is the same as "diagnosis". In addition to the early diagnosis of colorectal tumors, it also includes the diagnosis of the middle and late stages of colorectal tumors, and also includes colorectal tumor screening, risk assessment, prognosis, disease identification, and disease stages. Selection of diagnostic and therapeutic targets.

The application of colorectal tumor marker combination SDC2, COL4A1/COL4A2 and ITGA4 makes the early diagnosis of colorectal tumors possible. When it is determined that a gene methylated in a cancer cell is methylated in a clinically or morphologically normal cell, this indicates that the normal cell is developing into cancer. In this way, colorectal cancer can be diagnosed at an early stage by the methylation of the colorectal tumor-specific SDC2, COL4A1/COL4A2, and ITGA4 gene combination in normal appearance cells.

Among them, early diagnosis refers to the possibility of detecting cancer before metastasis, preferably before the morphological changes of tissues or cells can be observed.

In addition to the early diagnosis of colorectal tumors, the reagents/kits of this application are also expected to be used for colorectal tumor screening, risk assessment, prognostic diagnosis, disease identification, diagnosis of disease stages, and selection of therapeutic targets.

As an alternative embodiment of the disease stage, diagnosis can be made by measuring the degree of methylation of the combination of SDC2, COL4A1/COL4A2 and ITGA4 genes obtained from a sample through the progress of colorectal tumors in different stages or periods. By comparing the SDC2, COL4A1/COL4A2, and ITGA4 gene combination methylation degree of nucleic acids isolated from samples of each stage of colorectal cancer with one or one isolated from samples from intestinal tissues without abnormal cell proliferation The methylation degree of SDC2, COL4A1/COL4A2 and ITGA4 gene combination of multiple nucleic acids can detect the specific stage of colorectal tumor in the sample.

Generally speaking, CpG islands refer to regions rich in CpG dinucleotides, usually located in the promoter and its vicinity. The detection sites of methylation in this application include not only CpG islands, but also other regions such as CpG sites that are heterozygously methylated in the genome or intergenic regions, or isolated CpG sites.

The methylation joint detection reagent of the combination of SDC2, COL4A1/COL4A2 and ITGA4 genes may be a methylation detection reagent in the prior art. In the prior art, there are many methods to detect the methylation of the target gene, such as methylation-specific PCR (MSP), methylation-specific quantitative PCR (qMSP), and methylation-specific DNA binding protein PCR, quantitative PCR and DNA chips, methylation-sensitive restriction endonucleases, bisulfite sequencing or pyrosequencing, etc. In addition, other methylation detection methods can be introduced through patent US62007687. Each detection method has its corresponding reagents, which can be used in this application to detect the methylation of SDC2, COL4A1/COL4A2 and ITGA4 gene combinations.

In some embodiments, the primers and/or probes detect the methylation of each gene in the SDC2, COL4A1/COL4A2, and ITGA4 gene combination by quantitative Methylation-Specific PCR (qMSP).

In some specific embodiments of the present application, the SDC2 gene capture sequence in the methylation combined detection reagent provided in the present application includes any one of the following nucleotide sequences:

I. The nucleotide sequence shown in SEQ ID NO: 1 or 2 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% Or a sequence of at least 97% or 98% or at least 99%, or 100% identity; and

II. The complementary sequence of the sequence shown in I; and/or

The capture sequence for the methylation detection of the COL4A1/COL4A2 gene includes any one of the following nucleotide sequences:

III. The nucleotide sequence shown in SEQ ID NO: 6 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

IV. The complementary sequence of the sequence shown in III; and/or

The capture sequence for the methylation detection of the ITGA4 gene includes any one of the nucleotide sequences shown below:

V. The nucleotide sequence shown in SEQ ID NO: 10 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

VI. The complementary sequence of the sequence shown in V.

In some specific embodiments of this application, the upstream primer in the primers for methylation detection of the SDC2 gene provided in this application includes any one of the nucleotide sequences shown below:

VII. The nucleotide sequence shown in SEQ ID NO: 3 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

VIII. The complementary sequence of the sequence shown in VII; and/or

The downstream primer in the primer for methylation detection of the SDC2 gene contains any one of the following nucleotide sequences:

The nucleotide sequence shown in IX, SEQ ID NO: 4 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

X, the complementary sequence of the sequence shown in IX; and/or

The upstream primer in the primer for methylation detection of the COL4A1/COL4A2 gene contains any one of the following nucleotide sequences:

The nucleotide sequence shown in any one of XI, SEQ ID NO: 7 or SEQ ID NO: 18 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence; and

XII, the complementary sequence of the sequence shown in XI; and/or

The downstream primer in the primer for methylation detection of the COL4A1/COL4A2 gene contains any one of the following nucleotide sequences:

The nucleotide sequence shown in any of XIII, SEQ ID NO: 8 or SEQ ID NO: 19 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence; and

XIV, the complementary sequence of the sequence shown in XIII;

In some embodiments, the primer pair for methylation detection of the COL4A1/COL4A2 gene is shown in SEQ ID NO: 7 and SEQ ID NO: 8;

In some embodiments, the primer pair for methylation detection of the COL4A1/COL4A2 gene is shown in SEQ ID NO: 18 and SEQ ID NO: 19;

The upstream primer in the primer for methylation detection of ITGA4 gene contains any one of the following nucleotide sequences:

The nucleotide sequence shown in any one of XV, SEQ ID NO: 11 or SEQ ID NO: 24 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence; and

XVI, the complementary sequence of the sequence shown in XV; and/or

The downstream primer in the primer for methylation detection of ITGA4 gene contains any one of the following nucleotide sequences:

The nucleotide sequence shown in any one of XVII, SEQ ID NO: 12 or SEQ ID NO: 25 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence; and

XVIII, the complementary sequence of the sequence shown in XVII;

In some embodiments, the primer pair for methylation detection of the ITGA4 gene is shown in SEQ ID NO: 11 and SEQ ID NO: 12;

In some embodiments, the primer pair for methylation detection of the ITGA4 gene is shown in SEQ ID NO: 24 and SEQ ID NO: 25;

In some embodiments, the methylation detection probe of the SDC2 gene in the methylation detection reagent provided in the present application includes any one of the following nucleotide sequences:

The nucleotide sequence shown in XIX, SEQ ID NO: 5 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

XX, the complementary sequence of the sequence shown in XIX; and/or

The COL4A1/COL4A2 gene methylation detection probe contains any one of the following nucleotide sequences:

The nucleotide sequence shown in XXI, SEQ ID NO: 9 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

XXII, the complementary sequence of the sequence shown in XXI; and/or

The ITGA4 gene methylation detection probe contains any one of the following nucleotide sequences:

The nucleotide sequence shown in XXIII, SEQ ID NO: 13 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

XXIV, the complementary sequence of the sequence shown in XXIII.

In some embodiments, the nucleic acid probe further includes one or more of a label such as a radioisotope, a fluorescent group, a bioluminescent compound, a chemiluminescent compound, a metal chelate and an enzyme.

In some embodiments, the label of the probe is a fluorescent group, and these fluorescent groups include but are not limited to one or more of VIC, ROX, FAM, Cy5, HEX, TET, JOE, NED, and Texas Red.

In some embodiments, the probes of the SDC2, COL4A1/COL4A2, and ITGA4 genes are labeled with the same fluorophore, so that the sum of the methylation levels of these genes can be easily detected through a fluorescence channel, instead of Each gene needs to be detected with a different fluorescence channel, which reduces the complexity of the experiment.

The application also provides a kit for detecting tumors, including the methylation detection reagent.

In some specific embodiments of the present application, the kit provided by the present application includes: a first container, which contains a capture reagent; a second container, which contains a primer pair for amplification; and a third container, which contains a probe.

In some specific embodiments of this application, the kit provided in this application also includes the commonly used reagents in the kit, such as the commonly used conversion agent in qMSP, which is used to convert all unmethylated cytosine bases into uracil, The methylated cytosine base remains unchanged. The conversion agent is not particularly limited. The reagents reported in the prior art that can convert cytosine to uracil can be used, such as hydrazine salt, bisulfite and bisulfite (such as sodium metabisulfite, subsulfite). One or more of potassium bisulfate, cesium bisulfite, ammonium bisulfite, etc.). Another example is DNA polymerase, dNTPs, Mg ²⁺ ions and buffers commonly used in the amplification of COL4A1 gene.

In some specific embodiments, the kit includes: a first container, which contains a capture reagent; a second container, which contains a primer pair for amplification; and a third container, which contains a probe. The fourth container contains a conversion reagent for converting unmethylated cytosine.

In some embodiments, the kit further includes instructions.

In some embodiments, the kit further includes nucleic acid extraction reagents.

In some embodiments, the kit further includes a sampling device.

This application also provides the above-mentioned methylation detection reagents, kits, capture sequences, primers and/or probes in the preparation of reagents or kits for methylation detection, or the preparation of reagents or kits for detecting colorectal tumors. application.

The application also provides the application of the above-mentioned methylation detection reagents, kits, capture sequences, primers and/or probes in methylation detection, or applications in the detection of colorectal tumors.

This application also provides a tumor detection system, which includes the following components:

(1) SDC2, COL4A1/COL4A2 and ITGA4 gene methylation joint detection components:

(2) Data processing components;

(3) Result output component;

In some embodiments, the methylation detection component contains a methylation detection instrument;

In some embodiments, the methylation detection component further contains one or more of the methylation detection reagents, kits, capture sequences, primers, and probes.

In some embodiments, the methylation detection instrument includes one or more of a fluorescent quantitative PCR machine, a PCR machine, and a sequencer.

In some embodiments, the data processing component contains a data processing machine.

The data processing machine includes any equipment or instrument or device that can perform data processing that can be used by those skilled in the art.

In some embodiments, the data processing machine includes one or more of a calculator and a computer.

The computer is loaded with any software or program that can be used by those skilled in the art that can perform data processing or statistical analysis.

In some embodiments, the computer includes a computer loaded with one or more software of SPSS, SAS, and Excel.

In some embodiments, the result output member includes a result output device.

The output device includes any equipment or instrument or device that can display the data processing result as readable content.

In some embodiments, the result exporter includes one or more of a screen and a paper report.

In some embodiments, the data processor is configured to a. receive the test data of the test sample and the normal control sample; b. store the test data of the test sample and the normal control sample; c. compare the same type The test data of the test sample and the normal control sample; d. According to the comparison result, respond to the probability or possibility of the subject suffering from the tumor.

In some embodiments, the result output component is used to output the probability or likelihood that the subject has a tumor.

This application also provides a method for diagnosing colorectal tumors, which includes the following steps:

(1) Detect the methylation levels of SDC2, COL4A1/COL4A2 and ITGA4 genes in the test samples from the subject;

(2) Compare the SDC2, COL4A1/COL4A2 and ITGA4 gene methylation levels of the test sample and the normal control sample;

(3) Diagnose colorectal tumors based on the deviation of the methylation level of the test sample and the normal control sample.

The diagnostic method of the present application can be used before and after the treatment of colorectal tumors or in combination with the treatment of colorectal tumors, after treatment, such as evaluating the success of the treatment or monitoring the remission, recurrence and/or progress (including metastasis) of the colorectal tumor after treatment.

Another aspect of the present application provides a method for treating colorectal tumors, the method comprising administering surgery, chemotherapy, radiotherapy, radiotherapy and chemotherapy, immunotherapy, oncolytic virus therapy to patients diagnosed with colorectal tumors by the above diagnosis method , Or any other types of colorectal tumor treatment methods used in the art and combinations of these treatment methods.

In some embodiments, the data processing component in the tumor detection system, or the judgment standard of the colorectal tumor diagnosis method is: judging the tumor based on the cutoff value of the Ct value of methylation-specific quantitative PCR (qMSP) Specimen and normal specimens.

In some embodiments, the cutoff value of the Ct value is about 36-39.

In some embodiments, the cutoff value of the Ct value is about 38.

In some embodiments, the above-mentioned cutoff value of the Ct value is about 36-39 or the sample targeted for 38 is a stool sample.

In some embodiments, when the Ct value of the test sample is less than the threshold value of the Ct value, it is determined as a tumor sample, and when the Ct value of the test sample is greater than or equal to the threshold value of the Ct value, it is determined as Normal sample.

In some embodiments, the tumor described in this application is a colorectal tumor.

In some embodiments, the tumor described in this application is colorectal cancer or adenoma.

In some embodiments, the sample or sample type targeted by this application includes tissue, body fluid, or excrement.

In some embodiments, the tissue described herein comprises intestinal tissue.

In some embodiments, the body fluid described in this application includes blood, extracellular fluid, tissue fluid, lymph fluid, cerebrospinal fluid, or aqueous humor.

In some embodiments, the blood described in this application includes, serum, and plasma.

In some embodiments, the excrement comprises sputum, urine, saliva or feces.

In some embodiments, the excrement comprises feces.

In some embodiments, there is also provided a method for treating colorectal tumors, the method comprising the following steps:

(1) Combine the test sample derived from the subject with the primer of claim 1 or 2, or the capture sequence of claim 3 or 4, or the nucleic acid probe of claim 5 or 6, or claim 7. -9 contact with the kit of any of the reagents or the kit of claim 10 to detect the methylation level of the gene in the test sample;

(2) Compare the gene methylation level of the test sample with the normal control sample; and

(3) Diagnose colorectal tumors based on the deviation of the methylation level of the test sample and the normal control sample;

(4) Administration of anti-colorectal tumor drugs to subjects diagnosed with colorectal tumors.

In some embodiments, methylation-specific quantitative PCR (qMSP) is used to detect the methylation level of genes.

In some embodiments, the colorectal tumor sample and the normal sample are judged based on the cutoff value of the Ct value quantified by methylation fluorescence.

This application has discovered through research that in some specific embodiments, the methylation level of the promoter region of each gene in the SDC2, COL4A1/COL4A2 and ITGA4 gene combination can be detected by the primers, capture reagents, and nucleic acid probes. It is good to distinguish colorectal cancer specimens from stool specimens. And the detection sensitivity and specificity for bowel cancer are extremely high.

Compared with the existing markers for the detection of colorectal cancer, the primers, capture reagents, nucleic acid probes, SDC2, COL4A1/COL4A2 and ITGA4 gene combinations and technical solutions provided in this application can be detected with extremely high sensitivity and specificity For colorectal cancer, the detection sensitivity and specificity of the technical solution of the present application for colorectal cancer are higher than 90%. The specific points are as follows:

1. A technical solution of this application performs joint detection of methylation of SDC2, COL4A1/COL4A2 and ITGA4 gene combination. This method can realize joint detection of multiple genes, which greatly reduces the complexity of the test and improves the detection effectiveness.

2. In the above-mentioned another technical solution, the methylation detection reagent of the combination of SDC2, COL4A1/COL4A2 and ITGA4 genes can detect 91.36% of colorectal cancers in stool samples with a specificity of 95.28%, which can be easily Stool is used as a test sample to make a reliable diagnosis of colorectal cancer. The stool sample is very easy to obtain, the sampling is non-invasive and simple, and it will not cause any pain and inconvenience to the patient.

3. The above-mentioned another technical solution contains methylation detection reagents of SDC2, COL4A1/COL4A2 and ITGA4 gene combination. The extraction detection method can easily and accurately judge colorectal cancer and normal people. The methylation of this gene combination Chemical testing reagents are expected to be used in stool genetic testing kits and serve the clinical testing of bowel cancer.

4. The reagent/kit in the other technical solution mentioned above is to detect and diagnose cancer by methylation level. More and more studies have confirmed that methylation change is an early event in the process of tumorigenesis, and methylation is detected. Abnormalities are easier to find early lesions.

Description of the drawings

Figure 1 is the amplification curve of the primer pair S0 in Example 1;

Figure 2 is the amplification curve of the primer pair S0 in Example 1;

Figure 3 is an amplification curve of the primer pair S0 in Example 1;

Figure 4 is the amplification curve of the primer pair CO in Example 2;

Figure 5 is an amplification curve of the primer pair C1 in Example 2;

Figure 6 is the amplification curve of primer pair C2 in Example 2;

Figure 7 is the amplification curve of the primer pair I0 in Example 3;

Figure 8 is the amplification curve of the primer pair I1 in Example 3;

Figure 9 is the amplification curve of the primer pair I0 in Example 3;

Figure 10 shows the ROC curve of 935 stool specimens in Example 4 for the combined detection of SDC2, COL4A1/COL4A2 and ITG4 genes for colorectal cancer and adenomas (≧1cm);

Figure 11 shows the detection results of 23 cases of colorectal cancer patients with preoperative and postoperative stool samples detected by the method of combined detection of SDC2, COL4A1/COL4A2 and ITG4 genes in Example 5;

Figure 12 shows the ROC curve of 240 stool specimens in Comparative Example 1, SOX21 gene detection for colorectal cancer and adenoma;

Figure 13 shows the ROC curve of the combined detection of colorectal cancer with ITGA4, COL4A1/COL4A2 genes in 109 stool specimens in Comparative Example 2.

Detailed ways

The technical solutions of the present application are further described below through specific embodiments, and the specific embodiments do not represent a limitation on the protection scope of the present application. Some non-essential modifications and adjustments made by others based on the concept of this application still belong to the scope of protection of this application.

The "capture sequence", "primer" or "probe" in the present application refers to an oligonucleotide that contains a region complementary to a sequence of at least 6 consecutive nucleotides of a target nucleic acid molecule (for example, a target gene). In some embodiments, at least a portion of the sequence of the primer or probe is not complementary to the amplified sequence. In some embodiments, the primer or probe contains at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 relative to the target molecule. A region where the sequence of consecutive nucleotides is complementary. When a primer or probe contains a region "complementary to at least x consecutive nucleotides of the target molecule", the primer or probe is at least 95% of at least x consecutive or discontinuous block nucleotides of the target molecule Complementary. In some embodiments, the primer or probe is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, 96%, at least 97%, at least 98%, at least 99%, or 100% complementary.

In this application, "normal" samples refer to samples of the same type isolated from individuals known to be free of the cancer or tumor.

In this application, the "subject" is a mammal, such as a human.

The samples for methylation detection in this application include but are not limited to DNA, or RNA, or DNA and RNA samples containing mRNA, or DNA-RNA hybrids. The DNA or RNA can be single-stranded or double-stranded.

In this application, "methylation level" and "methylation degree" can usually be expressed as the percentage of methylated cytosine, which is the number of methylated cytosine divided by the number of methylated cytosine and the amount of unmethylated cytosine. The sum of the number of methylated cytosines; and the method of dividing the number of methylation target genes by the number of internal reference genes is generally used to express the methylation level; and other methods of expressing the methylation level in the prior art.

In this application, "sample" is the same as "specimen".

The term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and/or" means that any of the listed items can be used alone, or any combination of two or more of the listed items can be used . For example, if the composition, combination, structure, etc. are described as including (or including) components A, B, C and/or D, the composition may include A alone; B alone; C alone; D alone Contains the combination of A and B; contains the combination of A and C; contains the combination of A and D; contains the combination of B and C; contains the combination of B and D; contains the combination of C and D; contains the combination of A, B and C Combination; including A, B and D combination; including A, C and D combination; including B, C and D combination; or A, B, C and D in combination.

Example 1

According to Table 1, design SDC2 three pairs of primers to study the specificity of amplification.

Table 1 Primer of SDC2

The above 3 pairs of primers were used to detect DNA from stool samples of 2 patients with colorectal cancer and DNA from 2 normal human stool samples to analyze the specificity of amplification.

The amplification curve of SDC2 amplified by 3 pairs of primers is shown in Figure 1-3.

The results show that the SDC2 primer has a better amplification specificity for S0; while the amplification curves of the other two pairs of primers have little difference between negative and positive samples, and they cannot effectively distinguish between negative and positive samples.

Example 2

According to Table 2, three pairs of COL4A1/COL4A2 primers were designed to study the specificity of amplification.

Table 2 COL4A1/COL4A2 primers

The above 3 pairs of primers were used to detect the DNA of the stool samples of 3 patients with colorectal cancer and the DNA of 3 normal human stool samples to analyze the specificity of amplification.

The amplification curve of COL4A1/COL4A2 with 3 pairs of primers is shown in Figure 4-6.

The results showed that the COL4A1/COL4A2 primer had better amplification specificity for C0 and C1; while the primer pair C2 did not amplify COL4A1/COL4A2.

Example 3

According to Table 3, design ITGA4 three pairs of primers to study the specificity of amplification.

Table 3 Primer of ITGA4

The above 3 pairs of primers were used to detect the DNA of the stool samples of 3 patients with colorectal cancer and the DNA of 3 normal human stool samples to analyze the specificity of amplification.

The amplification curve of ITGA4 amplified by 3 pairs of primers is shown in Figure 7-9.

The results show that ITGA4 primer has good amplification specificity for I0 and I2; while the amplification curve of primer pair I1 has little difference between negative and positive samples, and it cannot effectively distinguish negative and positive samples.

Example 4

935 stool specimens (359 cases of colorectal cancer, 67 cases of adenoma (≧1cm), 509 cases of non-tumor individuals, all confirmed by colonoscopy or pathology) were selected for grinding and centrifugation, and 50ul capture magnetic beads (containing SDC2, COL4A2) were added. , ITGA4 and the capture sequence of the internal reference gene ACTB), and operate according to the technical scheme described below,

The technical solutions are as follows:

1) Collect stool specimens of normal people and colorectal tumor patients with colonoscopy pathology results, mix and grind 1g stool: 4mL protective solution, centrifuge at 5000rpm for 10min, take the supernatant and discard the precipitate;

2) Take out 10mL of supernatant and centrifuge again, take 3.2mL of supernatant, add 2mL of lysate and 50ul of capture magnetic beads M1, incubate at 95°C for 15min, and then place at room temperature for 30min;

3) Put a portion of the supernatant on a magnetic stand, wash off the magnetic beads and transfer to a 2mL centrifuge tube, add 800ul washing solution W1, incubate at 1300 rpm for 1 min at room temperature, put on the magnetic stand to aspirate the supernatant, repeat 2 times;

4) Add 50ul of eluent, incubate at 1300 rpm for 5 minutes at room temperature, place on a magnetic stand, and transfer the eluent to a new EP tube within 3 minutes;

5) Use EZ DNA Methylation Kit (Zymo Research) to methylate the DNA fragments in the previous step according to the method in Reference [2], and use 20ul of the final eluate for qMSP detection.

Finally, 20ul of Bisulfite-transformed DNA is obtained. Then perform qMSP detection, and finally determine the methylation level of the SDC2, COL4A1/COL4A2 and ITG4 gene combination in the specimen according to the CT value.

The qMSP reaction system of this example: 30ul (nuclease-free water 2.98ul, 5×Colorless GoTaq Flexi Buffer 6ul, MgCl ₂ (25mM) 5ul, dNTPs (10mM) 1ul, GoTaq Hot Start polymerase 0.6ul, ACTB-FP (100uM) 0.08ul, ACTB-RP (100uM) 0.08ul, ACTB-Probe (100uM) 0.06ul, SDC2-FP (100uM) 0.12ul, SDC2-RP (100uM) 0.12ul, SDC2-Probe (100uM) 0.04ul, COL4A2- FP (100uM) 0.06ul, COL4A2-RP (100uM) 0.06ul, COL4A2-Probe (100uM) 0.04ul, ITGA4-FP (100uM) 0.06ul, ITGA4-RP (100uM) 0.06ul, ITGA4-Probe (100uM) 0.04 ul, DNA 10ul). Reaction procedure: 95°C for 5 min, (95°C for 15s, 58°C for 30s, 72°C for 30s) × 48 Cycles, 40°C for 30s.

The capture and PCR reaction uses ACTB as the internal reference gene, and finally judges the methylation level in the specimen according to the CT value. The target gene is judged as positive with a CT value ≦ 38, and it is judged as negative with a CT value> 38.

Because the 5'ends of the COL4A1 and COL4A2 genes are close to each other, they are separated by 127bp, which is a shared bidirectional promoter region. The methylation region detected in this example is the bidirectional promoter region of the COL4A1 and COL4A2 genes. Therefore, in this embodiment, the gene direction of COL4A2 of the two genes is selected to annotate sequence information.

The methylation sites of SDC2, COL4A1/COL4A2 and ITGA4 genes are mainly located in the promoter region or nearby CpG islands.

In this example, the same fluorescent group is used to label the PCR probes of SDC2, COL4A2 and ITGA4, so the sum of the methylation levels of these genes can be easily detected through a fluorescent channel, without the need for each gene Using different fluorescence channels for detection reduces the complexity of the experiment.

The capture sequence and primer probe of this embodiment are as follows:

SEQ ID NO. 1: SDC2 capture sequence 1:

5’-AGCCCGCGCACACGAATCCGGAGCAGAGTACCG-3’ or

SEQ ID NO. 2: Capture sequence of SDC2 2:

5’-CTCCTGCCCAGCGCTCGGCGCAGCCCGC-3’

SDC2 qMSP primer probe:

SEQ ID NO.3: SDC2-FP: 5’-GAGGAAGCGAGCGTTTTC-3’

SEQ ID NO. 4: SDC2-RP: 5’-AAAATACCGCAACGATTACGA-3’

SEQ ID NO.5: SDC2-Probe: 5’-AGTTTCGAGTTCGAGTTTTCGAGTTTG-3’

SEQ ID NO.6: Capture sequence of COL4A1/COL4A2:

5’-GCTGCTGCCCGAACGCATTGGCCCTTCCAGAAGCA-3’

QMSP primer probe for COL4A1/COL4A2:

SEQ ID NO.7: COL4A1/COL4A2-FP: 5’-AGAGAGTTTAGTAAGGTCGGGC-3’

SEQ ID NO. 8: COL4A1/COL4A2-RP: 5’-GACTTCAAAAACTACTACCCG-3’

SEQ ID NO.9: COL4A1/COL4A2-Probe: 5’-TGTCGGTGTGTCGTCGGC-3’

SEQ ID NO.10: Capture sequence of ITGA4:

5’-CTACGCGCGGCTGCAGGGGGCGCTGGGGAACCT-3’

QMSP primer probe for ITGA4:

SEQ ID NO.11: ITGA4-FP: 5’-ACGCGAGTTTTGCGTAGAC-3’

SEQ ID NO.12: ITGA4-RP: 5’-GCTAAATAAAATCCCGAACG-3’

SEQ ID NO.13: ITGA4-Probe: 5’-ACGGAGTTCGGTTTTGCGTTTTC-3’

Based on the results of these 935 stool specimens, IBM SPSS statistics 20 software was used to draw the ROC curve of the marker combination for detecting colorectal cancer and adenoma, as shown in Figure 9.

The results showed that the combined detection of SDC2, COL4A1/COL4A2 and ITG4 gene methylation has a sensitivity of 91.36% for colorectal cancer and a sensitivity of 50.75% for adenoma when the specificity is 95.28%; SDC2, COL4A1/COL4A2 and Combined detection of ITG4 gene methylation, the area under the ROC curve for colorectal cancer was 0.979, 95% CI was 0.970 to 0.988, the area under the ROC curve for adenoma was 0.832, and 95% CI was 0.771 to 0.894.

Example 5

Select 23 patients diagnosed with colorectal cancer, collect patient's stool samples before and 3-6 months after operation, and use the marker combination (SDC2, COL4A1/COL4A2 and ITGA4) of this application to detect the preoperative and surgical After a total of 46 stool specimens, the methylation level of the marker combination in the stool before and after surgery was compared.

The detection process and result judgment criteria of the marker combination are the same as in Example 4. Target gene CT value ≦ 38 is judged as positive, CT value> 38 is judged as negative. The result is shown in Figure 10.

The results showed that the test results of these 23 patients with colorectal cancer were positive before the operation, and the stool specimens were tested again after the operation, and the results were all negative. It shows that the methylation level of the marker combination is significantly reduced after the tumor is surgically removed, suggesting that the marker combination can be used for postoperative follow-up monitoring of patients with colorectal cancer.

Comparative example 1

The study found that the detection of methylated SOX21 gene in feces can be used for the auxiliary diagnosis of colorectal cancer, and the detection of colorectal cancer in feces is now used as a comparative example. The following is the procedure and results of detecting SOX21 gene in feces.

Select 240 stool specimens (80 cases of colorectal cancer, 77 cases of ≧1cm adenoma, 83 cases of normal, all confirmed by colonoscopy or pathology), ground and centrifuge, and add 50ul capture magnetic beads (containing SOX21 and internal reference gene ACTB capture Sequence), and operate according to the "technical solution" described in Example 4, and finally obtain 20ul of Bisulfite-transformed DNA. Then perform qMSP detection, and finally determine the SOX21 methylation level in the specimen according to the CT value.

The capture sequences, primer probes, and qMSP reaction system and procedures contained in the reagents can be found in reference [2]. Based on the results of 240 stool specimens, the ROC curve of SOX21 gene detection for colorectal cancer and adenoma was drawn, as shown in Figure 11.

The results showed that when the specificity of methylated SOX21 was 97.6%, the sensitivity to colorectal cancer was 80%, and the sensitivity to adenoma was 33.8%; the area under the ROC curve for SOX21 detection of colorectal cancer was 0.926, 95% The CI was 0.885～0.968, the area under the ROC curve for detecting adenoma was 0.667, and the 95% CI was 0.583～0.751.

Comparative example 2

Select 161 stool specimens (79 cases of colorectal cancer, 82 cases of normal, all confirmed by colonoscopy or pathology), grinding and centrifugation, adding 50ul capture magnetic beads (containing SOX21, SDC2 and internal reference gene ACTB capture sequence), and press The "technical solution" operation described in Example 4 finally obtained 20ul of Bisulfite-transformed DNA. Then perform qMSP detection, and finally determine the methylation level of SOX21 and SDC2 in the specimen according to the CT value.

The capture sequence, primer probe, and qMSP reaction system and procedures of SOX21 are the same as those in Comparative Example 1; the capture sequence, primer probe, and qMSP reaction system and procedures of SDC2 are the same as those in Example 4.

The test results showed that the sensitivity of methylated SDC2 gene detection was 88.6%, the specificity was 93.9%; the area under the curve was 0.939. The detection sensitivity of methylated SOX21 gene is 79.7%, the specificity is 86.6%; the area under the curve is 0.924. The detection sensitivity of methylated SOX21 gene and methylated SDC2 gene combination was 87.34%, and the specificity was 93.9%. The detection sensitivity of the combined detection of methylated SDC2 gene and methylated SOX21 gene is lower than the detection sensitivity of methylated SDC2 gene alone as a colorectal cancer marker, and it is also lower than that of the application of SDC2, COL4A1/COL4A2 and ITG4 gene A Based on the sensitivity of combined detection.

Comparative example 3

Select 109 stool specimens (61 cases of colorectal cancer, 48 cases are normal, all confirmed by colonoscopy or pathology), ground and centrifuge, and add 50ul capture magnetic beads (containing the capture sequence of ITGA4, COL4A1/COL4A2 and internal reference gene ACTB)

The capture sequence of ITGA4, COL4A1/COL4A2, primer probes, and qMSP reaction system and procedures are the same as in Example 4.

Based on the results of these 109 stool samples, the ROC curve of the marker combination for colorectal cancer detection was drawn, as shown in Figure 12.

The results showed that the area under the curve distinguishing colorectal cancer from normal samples was 0.964. When the positive judgment Ct value is 38, the sensitivity of methylated ITGA4+COL4A1/COL4A2 gene for colorectal cancer detection is 86.89%, and the specificity is 91.67%. Its sensitivity and specificity are lower than the sensitivity and specificity of the combined detection of SDC2, COL4A1/COL4A2 and ITG4 gene methylation in this application.

The above are only the preferred embodiments of this application. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of this application, several improvements and modifications can be made, and these improvements and modifications are also Should be regarded as the scope of protection of this application.

references:

1. Fischer G, Schmidt, Cornelia, et al. Identification of a novel sequence element in the common promoter region of human collagen type IV genes, involved in the regulation of divergent prescription.Biochem.J.1993 292:687-695.

2. Niu F, Wen J, Fu X, et al. Stool DNA Test of Methylated Syndecan-2 for the Early Detection of Colorectal Neoplasia. Cancer Epidemiol Biomarkers Prev 2017; 26:1411-1419.

3. Liu Xianglin, Wen Jialing, Niu Feng, et al. Application value of detection of SOX21 gene methylation in feces in the diagnosis of colorectal cancer[J]. Chinese Journal of Cancer Prevention and Treatment,2018(24):1710-1715.

Claims (15)

1. A primer comprising the same as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14. SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and their complementary sequences have at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% Or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or at least any one of the sequences with 100% identity.
2. The primer according to claim 1, which contains the same as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID NO: 25 have at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% Or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or any one of the sequences with 100% identity;

   Preferably, the primers include SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 18 And SEQ ID NO: 19; and SEQ ID NO: 24 and SEQ ID NO: 25 have at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or at least one primer pair in a sequence that is 100% identical;

   Preferably, the primers include SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 7 and SEQ ID NO: 8; and SEQ ID NO: 11 and SEQ ID NO: 12 as shown in the primer pair;

   Preferably, the primers include SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 18 and SEQ ID NO: 19; and SEQ ID NO: 24 and SEQ ID NO: 25.
3. A capture sequence comprising the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, and their complementary sequence having at least 85% or at least 90 % Or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or at least any one of the sequences of 100% identity.
4. The capture sequence according to claim 3, which contains at least 85% or at least 90% of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10 Or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or at least one sequence of 100% identity;

   Preferably, the capture sequence includes the sequence shown in SEQ ID NO: 1, SEQ ID NO: 6 and SEQ ID NO: 10;

   Preferably, the capture sequence includes the sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10.
5. A nucleic acid probe comprising the sequence shown in SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, and their complementary sequence having at least 85% or at least 90% or at least 91 % Or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or at least any one of the sequences of 100% identity.
6. The nucleic acid probe according to claim 5, which comprises at least one of SEQ ID NO: 5, SEQ ID NO: 9 and SEQ ID NO: 13;

   Preferably, the nucleic acid probe comprises the sequence shown in SEQ ID NO: 5, SEQ ID NO: 9 and SEQ ID NO: 13.
7. A detection reagent, comprising the primer according to claim 1 or 2, or the capture sequence according to claim 3 or 4, or the nucleic acid probe according to claim 5 or 6.
8. The multi-gene methylation combined detection reagent according to claim 7, comprising capture sequences, primers and/or probes for SDC2, COL4A1/COL4A2 and ITGA4 genes.
9. The multi-gene methylation combined detection reagent according to claim 7, wherein:

   The capture sequence for the methylation detection of the SDC2 gene includes any one of the nucleotide sequences shown below:

   I. The nucleotide sequence shown in SEQ ID NO: 1 or 2 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% Or a sequence of at least 97% or 98% or at least 99%, or 100% identity; and

   II. The complementary sequence of the sequence shown in I; and/or

   The capture sequence for the methylation detection of the COL4A1/COL4A2 gene includes any one of the following nucleotide sequences:

   III. The nucleotide sequence shown in SEQ ID NO: 6 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

   IV. The complementary sequence of the sequence shown in III; and/or

   The capture sequence for the methylation detection of the ITGA4 gene includes any one of the nucleotide sequences shown below:

   V. The nucleotide sequence shown in SEQ ID NO: 10 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

   VI. The complementary sequence of the sequence shown in V;

   Preferably, the upstream primer in the primers for methylation detection of the SDC2 gene contains any one of the following nucleotide sequences:

   VII. The nucleotide sequence shown in SEQ ID NO: 3 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

   VIII. The complementary sequence of the sequence shown in VII; and/or

   The downstream primer in the primer for methylation detection of the SDC2 gene contains any one of the following nucleotide sequences:

   The nucleotide sequence shown in IX, SEQ ID NO: 4 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

   X, the complementary sequence of the sequence shown in IX; and/or

   The upstream primer in the primer for methylation detection of the COL4A1/COL4A2 gene contains any one of the following nucleotide sequences:

   The nucleotide sequence shown in any one of XI, SEQ ID NO: 7 or SEQ ID NO: 18 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence; and

   XII, the complementary sequence of the sequence shown in XI; and/or

   The downstream primer in the primer for methylation detection of the COL4A1/COL4A2 gene contains any one of the following nucleotide sequences:

   The nucleotide sequence shown in any of XIII, SEQ ID NO: 8 or SEQ ID NO: 19 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence; and

   XIV, the complementary sequence of the sequence shown in XIII;

   Preferably, the primer pair for the methylation detection of the COL4A1/COL4A2 gene is shown in SEQ ID NO: 7 and SEQ ID NO: 8; and

   Preferably, the primer pair for the methylation detection of the COL4A1/COL4A2 gene is shown in SEQ ID NO: 18 and SEQ ID NO: 19; and/or

   The upstream primer in the primer for methylation detection of ITGA4 gene contains any one of the following nucleotide sequences:

   The nucleotide sequence shown in any one of XV, SEQ ID NO: 11 or SEQ ID NO: 24 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence; and

   XVI, the complementary sequence of the sequence shown in XV; and/or

   The downstream primer in the primer for methylation detection of ITGA4 gene contains any one of the following nucleotide sequences:

   The nucleotide sequence shown in any one of XVII, SEQ ID NO: 12 or SEQ ID NO: 25 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence; and

   XVIII, the complementary sequence of the sequence shown in XVII;

   Preferably, the primer pair for the methylation detection of the ITGA4 gene is shown in SEQ ID NO: 11 and SEQ ID NO: 12;

   Preferably, the primer pair for methylation detection of the ITGA4 gene is shown in SEQ ID NO: 24 and SEQ ID NO: 25;

   Preferably, the probe for methylation detection of the SDC2 gene contains any one of the following nucleotide sequences:

   The nucleotide sequence shown in XIX, SEQ ID NO: 5 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

   XX, the complementary sequence of the sequence shown in XIX; and/or

   The COL4A1/COL4A2 gene methylation detection probe contains any one of the following nucleotide sequences:

   The nucleotide sequence shown in XXI, SEQ ID NO: 9 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

   XXII, the complementary sequence of the sequence shown in XXI; and/or

   The ITGA4 gene methylation detection probe contains any one of the following nucleotide sequences:

   The nucleotide sequence shown in XXIII, SEQ ID NO: 13 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence; and

   XXIV, the complementary sequence of the sequence shown in XXIII.
10. A kit comprising the primer of claim 1 or 2, or the capture sequence of claim 3 or 4, or the nucleic acid probe of claim 5 or 6, or the reagent of any one of claims 7-9;

    Preferably, the kit includes: a first container, which contains a capture reagent; a second container, which contains a primer pair for amplification; and a third container, which contains a probe.
11. The primer of claim 1 or 2, or the capture reagent of claim 3 or 4, or the nucleic acid probe of claim 5 or 6, or the reagent of any one of claims 7-9, when preparing methylation Application in reagents or kits for detection, or preparation of reagents or kits for detection of colorectal tumors.
12. The primer of claim 1 or 2, or the capture reagent of claim 3 or 4, or the nucleic acid probe of claim 5 or 6, or the reagent of any one of claims 7-9, or the capture reagent of claim 10. The kit can be used in methylation detection or in colorectal tumor detection.
13. The use according to any one of claims 11 or 12, wherein the colorectal tumor is colorectal cancer or adenoma.
14. The application according to any one of claims 11 or 12, wherein the sample to be tested for detection includes tissue, body fluid or excrement;

    Preferably, the tissue comprises intestinal tissue;

    Preferably, the body fluid comprises blood, extracellular fluid, tissue fluid, lymph fluid, cerebrospinal fluid or aqueous humor;

    Preferably, the blood contains serum and plasma;

    Preferably, the excrement contains sputum, urine, saliva or feces;

    Preferably, the excrement contains feces.
15. A method for treating colorectal tumors, said method comprising the following steps:

    (1) Combine the test sample derived from the subject with the primer of claim 1 or 2, or the capture sequence of claim 3 or 4, or the nucleic acid probe of claim 5 or 6, or claim 7. -9 contact with the kit of any of the reagents or the kit of claim 10 to detect the methylation level of the gene in the test sample;

    (2) Compare the gene methylation level of the test sample with the normal control sample; and

    (3) Diagnose colorectal tumors based on the deviation of the methylation level of the test sample and the normal control sample;

    (4) To administer anti-colorectal tumor drugs to subjects diagnosed with colorectal tumors;

    Preferably, methylation-specific quantitative PCR (qMSP) is used to detect the methylation level of genes;

    Preferably, the colorectal tumor sample and the normal sample are judged according to the boundary value of the Ct value of the methylation fluorescence quantitative;

    Preferably, the boundary value of the Ct value ranges from about 36 to 39; more preferably, the boundary value of the Ct value is about 38;

    Preferably, when the Ct value of the test sample is less than the threshold value of the Ct value, it is determined as a colorectal tumor sample, and when the Ct value of the test sample is greater than or equal to the threshold value of the Ct value, it is determined as normal specimen;

    Preferably, the test sample targeted by the method includes tissue, body fluid or excrement;

    Preferably, the tissue is intestinal tissue;

    Preferably, the body fluid comprises blood, extracellular fluid, tissue fluid, lymph fluid, cerebrospinal fluid or aqueous humor;

    Preferably, the blood contains serum and plasma;

    Preferably, the excrement sputum, urine, saliva or feces;

    More preferably, the excrement is feces.

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