WO2019223520A1 - Tumor marker, methylation detection reagent, kit and application thereof - Google Patents

Abstract

Disclosed in the present invention are a tumor marker, a methylation detection reagent, a kit and an application thereof. A colorectal cancer specimen may be distinguished from a normal human fecal specimen by means of detecting the methylation level of a TLX2 gene promoter region or jointly detecting the methylation levels of COL4A2 and TLX2 gene promoter regions. The present invention utilizes a methylation detection reagent containing a TLX2 gene or the two genes of COL4A2 and TLX2 to detect colorectal cancer.

Description

Tumor marker, methylation detection reagent, kit and application thereof Technical field

The invention relates to the field of biotechnology, in particular to tumor markers, methylation detection reagents, kits and applications thereof.

Background technique

Colorectal cancer, also known as colorectal cancer, is a common malignant tumor of the digestive tract. Its incidence has been increasing year by year in China. In some coastal areas of China, such as Shanghai and Guangzhou, the incidence of colorectal cancer has jumped to the second place, behind lung cancer. The formation of bowel cancer is currently thought to be the result of the accumulation of genetic defects and epigenetic defects. The early onset of colorectal cancer is insidious and often without obvious symptoms. In the later stage, symptoms such as blood in the stool, abdominal pain, and diarrhea may occur. And when symptoms appear, it is often late, which brings great pain and expensive treatment costs to patients. 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 include occult blood test and colonoscopy. The occult blood test has the problems of being easily affected by food or having a low detection rate of adenomas. Colonoscopy is the gold standard for the diagnosis of bowel cancer, but it is not highly compliant when used as a screening tool. Therefore, a highly accurate and compliant screening method for bowel cancer is urgently needed.

于2016年纳入美国的肠癌筛查指南。该方法具有方便、无创、对肠癌和癌前病变腺瘤的检出率高等特点。要做成对肠癌检测高性能的粪便基因检测试剂盒，主要需要克服两大障碍：粪便DNA的提取和标志物选择。一方面，粪便中成分复杂，对下游反应的抑制物多，还有许多细菌DNA，要从这样的混合物中提取出人的目标基因，需要一套高敏的基因提取和纯化方法；另一方面，目前和肠癌相关的标志物很多，尤其是DNA甲基化标志物，因为研究表明，DNA甲基化是肿瘤形成的早期事件。但很多甲基化标志物在细胞、组织层面表现很好，当用于粪便、血液等筛查媒介时，其对肠癌的敏感性和特异性就降下来了，比如vimentin基因，其在组织中的敏感性有83％， 在粪便标本中就降到了46％(J Natl Cancer Inst.2005Aug 3；97(15):1124-32.)，类似的还有SFRP1、SFRP2、CDH4等基因，这样的标志物无法满足真正用于肠癌临床检测的需求。因此，挑选在粪便中对肠癌有极高检测敏感性和特异性的标志物或标志物组合是肠癌粪便基因检测的关键，并且这样的标志物有望真正用于肠癌的临床检测。 As a new method of screening for bowel cancer, stool gene testing is receiving increasing attention. this method

Included in the U.S. colon cancer screening guidelines in 2016. This method is convenient, non-invasive, and has a high detection rate for bowel cancer and precancerous adenomas. To make a high-performance stool gene test kit for colon 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 high-sensitivity gene extraction and purification method is required; on the other hand, There are many markers related to bowel cancer, especially DNA methylation markers, because research shows that DNA methylation is an early event of tumor formation. However, many methylation markers perform well at the cell and tissue levels. When used for screening media such as stool and blood, their sensitivity and specificity to bowel cancer are reduced, such as the vimentin gene, which The sensitivity in 83% was reduced to 46% in stool samples (J Natl Cancer Inst. 2005Aug 3; 97 (15): 1124-32.), And there were similar genes such as SFRP1, SFRP2, CDH4, etc. The markers cannot meet the needs for clinical detection of colon cancer. Therefore, the selection of markers or combinations of markers that have extremely high sensitivity and specificity for detecting bowel cancer in stool is the key to the detection of bowel cancer stool genes, and such markers are expected to be truly used for clinical detection of bowel cancer.

Summary of the Invention

In view of this, the present invention provides a tumor marker, capture sequence, primer pair, probe, methylation detection reagent, kit and application thereof. The tumor marker combination has extremely high sensitivity and specificity for colon cancer in stool.

Another object of the present invention is to provide a marker, a capture sequence, a primer pair, a probe, a methylation detection reagent, a kit and a method for non-invasively detecting tumors. In order to achieve the above-mentioned object, the present invention provides the following technical solutions:

The invention provides the application of the TLX2 gene and / or the COL4A2 gene in preparing a tumor marker combination.

In some specific embodiments of the present invention, the sequence of the TLX2 gene has at least 97.8%, at least 98.9%, at least 99.9%, or 100% identity with the sequence shown in Genebank Accession No. NC_000002.12;

In some specific embodiments of the present invention, the sequence of the COL4A2 gene has at least 97.8%, at least 98.9%, at least 99.9%, or 100% identity with the sequence shown in Genebank Accession No. NC_000013.11.

In some specific embodiments of the invention, the tumor is a colorectal tumor.

In some embodiments of the invention, the tumor is colorectal cancer or adenoma.

In some specific embodiments of the present invention, the sample to be tested is tissue, body fluid or excrement.

In some embodiments of the invention, the tissue is intestinal tissue.

In some embodiments of the invention, body fluids include, but are not limited to, blood, serum, plasma, extracellular fluid, interstitial fluid, lymph fluid, cerebrospinal fluid, or aqueous humor.

In some embodiments of the present invention, the excreta is sputum, urine or feces.

The invention also provides the application of the methylation detection reagent of the TLX2 gene and / or the COL4A2 gene in the preparation of a tumor detection reagent or a kit.

The methylation detection reagent of the TLX2 gene and / or the COL4A2 gene may be a methylation detection reagent in the prior art. In the prior art, there are various methods for detecting methylation of a target gene, such as Methylation-specific PCR (MSP), methylation-specific quantitative PCR (qMSP), PCR of methylated DNA-specific binding proteins, quantitative PCR and DNA chips, methylation-sensitive restriction enzymes, heavy sub Sulphate sequencing or pyrosequencing, etc. Each detection method has its corresponding reagent, and these reagents can be used to detect methylation of the COL4A2 and TLX2 gene genes by using the present invention.

The invention also provides a capture sequence, which has any one of the nucleotide sequences shown below:

XVII, having the nucleotide sequence shown in SEQ ID NO: 5;

XVIII. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 5 or a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5 Functionally similar nucleotide sequences obtained from CpG islands;

XIX, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 5 or having the core shown in SEQ ID NO: 1 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

XX, the complement of a sequence shown as XVII, XVIII or XIX.

The present invention also provides a primer pair, and the upstream primer has any one of the nucleotide sequences shown below:

XXI, having the nucleotide sequence shown in SEQ ID NO: 6;

XXII. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 6;

XXIII, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 6 or having the core shown in SEQ ID NO: 6 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

XXIV, the complement of a sequence shown by XXI, XXII or XXIII;

The downstream primer has any of the nucleotide sequences shown below:

XXV, having the nucleotide sequence shown in SEQ ID NO: 7;

XXVI, a nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 7;

XXVII. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 7 or having the core shown in SEQ ID NO: 7 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

XXVIII, the complement of a sequence shown by XXV, XXVI or XXVII.

The present invention also provides a probe having any one of the nucleotide sequences shown below:

XXIX, having the nucleotide sequence shown in SEQ ID NO: 8;

XXX. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 8;

XXXI, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 8 or having the core shown in SEQ ID NO: 8 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

XXXII, the complement of the sequence shown by XXIX, XXX or XXXI.

The present invention also provides a methylation detection reagent for the COL4A2 gene and / or TLX2 gene, including capture sequences, primers and / or probes for the COL4A2 gene and / or TLX2 gene.

In some specific embodiments of the present invention, capture sequences, primers, and / or probes obtained for CpG islands of the COL4A2 gene and / or the TLX2 gene are included.

In some specific embodiments of the present invention, the primers and / or probes detect methylation of the COL4A2 gene and / or the TLX2 gene by quantitative methylation-specific PCR (qMSP).

In some specific embodiments of the present invention, the methylation detection reagent provided by the present invention detects methylation of the COL4A2 gene and / or TLX2 gene genome, intergenic region or promoter region and the region near the promoter region. Level.

The presence of methylation in tumor tissue is considered to be an apparent modification of DNA with potential clinical value. There are methylations in the genome, intergenic regions, promoters, or nearby regions, and methylation in these regions may be related to tumors. At present, it has been confirmed in a variety of tumors that abnormal methylation of CpG islands in the promoter region of oncogenes or nearby regions results in inactivation of transcription. In some specific embodiments of the present invention, the methylation detection reagent includes a capture sequence, a primer, and / or a CpG island obtained from a promoter region of a COL4A2 gene and / or a TLX2 gene or a region near the promoter region. Probe.

In some specific embodiments of the present invention, the capture sequence of the COL4A2 gene in the methylation detection reagent has any one of the nucleotide sequences shown below:

I. It has the nucleotide sequence shown in SEQ ID NO: 1;

Ⅱ. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 1 Functionally similar nucleotide sequences obtained from CpG islands;

III. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 1 or having the core shown in SEQ ID NO: 1 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

IV. Complement to the sequence shown in I, II or III.

In some specific embodiments of the present invention, the upstream primer of the COL4A2 gene in the methylation detection reagent has any one of the nucleotide sequences shown below:

Ⅴ. It has the nucleotide sequence shown in SEQ ID NO: 2;

Ⅵ. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 2;

Ⅶ A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 2 or having a core shown in SEQ ID NO: 2 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

Ⅷ, the complement of the sequence shown in Ⅴ, Ⅵ or Ⅶ;

In some specific embodiments of the present invention, the downstream primer of the COL4A2 gene in the methylation detection reagent has any one of the nucleotide sequences shown below:

(Ii) having the nucleotide sequence shown in SEQ ID NO: 3;

X. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 3;

Ⅺ A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 3 or having the core shown in SEQ ID NO: 3 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

VII, the complement of a sequence such as IX, X or IX.

In some specific embodiments of the present invention, the probe of the COL4A2 gene in the methylation detection reagent has any one of the nucleotide sequences shown below:

XIII, having the nucleotide sequence shown in SEQ ID NO: 4;

XIV. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 4;

XV, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 4 or having the core shown in SEQ ID NO: 4 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

XVI, the complement of the sequence shown by XIII, XIV or XV.

In some specific embodiments of the present invention, the capture sequence of the TLX2 gene in the methylation detection reagent has any one of the nucleotide sequences shown below:

XVII, having the nucleotide sequence shown in SEQ ID NO: 5;

XVIII. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 5 or a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5 Functionally similar nucleotide sequences obtained from CpG islands;

XIX, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 5 or having the core shown in SEQ ID NO: 1 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

XX, the complement of a sequence shown as XVII, XVIII or XIX.

In some specific embodiments of the present invention, the upstream primer of the TLX2 gene in the methylation detection reagent has any one of the nucleotide sequences shown below:

XXI, having the nucleotide sequence shown in SEQ ID NO: 6;

XXII. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 6;

XXIII, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 6 or having the core shown in SEQ ID NO: 6 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

XXIV, the complement of a sequence shown by XXI, XXII or XXIII;

In some specific embodiments of the present invention, the downstream primer of the TLX2 gene in the methylation detection reagent has any one of the nucleotide sequences shown below:

XXV, having the nucleotide sequence shown in SEQ ID NO: 7;

XXVI, a nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 7;

XXVII. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 7 or having the core shown in SEQ ID NO: 7 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

XXVIII, the complement of a sequence shown by XXV, XXVI or XXVII.

In some specific embodiments of the present invention, the probe of the TLX2 gene in the methylation detection reagent has any one of the nucleotide sequences shown below:

XXIX, having the nucleotide sequence shown in SEQ ID NO: 8;

XXX. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 8;

XXXI, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 8 or having the core shown in SEQ ID NO: 8 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;

XXXII, the complement of the sequence shown by XXIX, XXX or XXXI.

The invention also provides a kit for detecting tumors, which comprises the capture sequence, primer pairs, probes or methylation detection reagents.

In some specific embodiments of the present invention, the kit provided by the present invention comprises: one or more containers divided into receiving reagents therein.

In some specific embodiments of the present invention, the kit provided by the present invention includes: a first container containing a capture sequence; a second container containing a primer pair for amplification; and a third container containing a probe.

In some specific embodiments of the present invention, the kit provided by the present invention further includes a common reagent in the kit, such as a transforming agent commonly used in qMSP, for converting unmethylated cytosine bases to uracil, The methylated cytosine bases remain unchanged. The conversion agent includes, but is not limited to, bisulfite, bisulfite or hydrazine, and the like. Another example is DNA polymerases, dNTPs, Mg ²⁺ ions and buffers commonly used in the amplification of COL4A2 and TLX2 genes.

The invention also provides the application of the aforementioned capture sequences, primer pairs, probes, methylation detection reagents, and kits in detecting tumors.

The invention also provides a tumor detection method, which distinguishes normal samples from tumor samples by detecting the methylation level of the COL4A2 gene and / or TLX2 gene.

In some specific embodiments of the present invention, it includes the following steps:

(1) detecting the methylation level of COL4A2 gene and / or TLX2 gene in a subject;

(2) comparing the methylation level of the subject's COL4A2 gene and / or TLX2 gene with that of a normal control sample;

(3) According to an increase in the methylation level of the TCOL4A2 gene and / or TLX2 gene of the subject compared to the methylation level of a normal control sample, it indicates that the subject has or is at risk of developing a tumor To distinguish between normal and tumor samples.

In some specific embodiments of the present invention, the present invention detects the methylation level of the COL4A2 gene and / or the TLX2 gene gene body, intergenic region or promoter region and the region near the promoter region.

In some specific embodiments of the present invention, a normal sample and a tumor sample are distinguished by detecting methylation levels in the promoter region of the COL4A2 gene and / or the TLX2 gene and a region near the promoter region.

In some specific embodiments of the present invention, the methylation level is determined by methylation-specific PCR, or methylation-specific quantitative PCR (qMSP), or PCR or quantitative PCR of methylated DNA-specific binding proteins. , And DNA chips, or methylation-sensitive restriction enzymes, or bisulfite sequencing, or pyrosequencing.

In some embodiments of the invention, the methylation level is detected by methylation-specific quantitative PCR (qMSP).

In some specific embodiments of the present invention, the methylation level is detected using the capture sequence, primer pair, probe, methylation detection reagent or the kit.

In some specific embodiments of the present invention, in step (1), detecting the methylation level of the subject's COL4A2 gene and / or TLX2 gene comprises the following steps:

a) Use the magnetic bead capture method to extract the DNA of the sample to be tested;

b) the DNA of the test sample is transformed with bisulfite, bisulfite or hydrazine;

c) methylation-specific quantitative PCR (qMSP) detection;

Preferably, extracting the DNA of the sample to be tested by the magnetic bead capture method includes the following steps;

Take the sample to be tested, mix and grind it in the protection solution, centrifuge, and take the supernatant;

Supernatant was centrifuged, the supernatant was taken, lysate and magnetic beads with specific complementary oligonucleotide capture sequences were added to the supernatant and incubated;

After discarding the supernatant, the magnetic beads were washed and transferred to a clean centrifuge tube, and the washing solution was added, and incubated at room temperature at 100-2000 rpm for 0.5-5min. The supernatant was placed on a magnetic stand and the supernatant was repeated 3 times;

The target gene DNA was eluted with a buffer.

In some specific embodiments of the present invention, the detection and interpretation criteria are: two genes of the COL4A2 gene and / or the TLX2 gene are jointly detected, and when at least one gene has a positive test result, the interpretation result is a tumor specimen; when the two genes are When the test results are negative, the interpretation is normal. Tumor specimens and normal specimens are judged according to the cutoff value of the predicted Ct value in the joint diagnosis of the two genes. The cutoff value of the Ct value in the stool specimen of the COL4A2 gene is 30 to 40. Preferably, the cutoff value of the Ct value in the stool specimen of the COL4A2 gene is preferred. The value is 34, the Ct value in the COL4A2 stool sample is less than the Ct value, and the Ct value in the COL4A2 stool sample is greater than or equal to the Ct value. The Ct value is negative; the Ct in the TLX2 stool sample is negative The cutoff value of the value is 32 to 42, preferably, the cutoff value of the Ct value in the TLX2 gene stool sample is 38, the cutoff value of the Ct value in the TLX2 gene stool sample is less than the cutoff value of the Ct value, and the cutoff value in the TLX2 gene stool sample is positive. A cutoff value of Ct value greater than or equal to the Ct value is negative. This threshold can be adjusted according to the actual situation.

The invention also provides a tumor detection system, the system includes the following components;

(1) The methylation detection component of the COL4A2 gene and / or the TLX2 gene;

(2) data processing components;

(3) result output component;

In some specific embodiments of the present invention, the methylation detection component contains one or more of a fluorescent quantitative PCR instrument, a PCR instrument, and a sequencer;

In some specific embodiments of the present invention, the methylation detection component further comprises the capture sequence, a primer pair, a probe, a methylation detection reagent or a kit.

In some specific embodiments of the present invention, the data processing component is configured to: a. Receive test data of a test sample and a normal control sample; b. Store test data of the test sample and a normal control sample; c. Test data on the same type of test sample and normal control sample; d. According to the comparison result, respond to the probability or possibility of the tumor of the test subject.

In some specific embodiments of the present invention, the result output component is used to output the probability or possibility that the test subject has a tumor.

In some specific embodiments of the present invention, the judgment standard of the data processing component is:

The COL4A2 gene and / or TLX2 gene are jointly tested. When at least one gene is positive, the interpretation result is a tumor specimen; when both genes are negative, the interpretation result is a normal specimen;

The detection result is positive or negative according to the cutoff value of the Ct value of the two genes. The cutoff value of the Ct value in the COL4A2 gene stool sample is 30 to 40. Preferably, the cutoff value of the Ct value in the COL4A2 gene stool sample is 34. The Ct value in the COL4A2 gene stool specimen is less than the cutoff value of the Ct value is positive, and the Ct value in the COL4A2 gene stool specimen is greater than or equal to the cutoff value of the Ct value;

The threshold value of the Ct value in the TLX2 gene stool sample is 32 to 42, preferably, the threshold value of the Ct value in the TLX2 gene stool sample is 38, and the Ct value in the TLX2 gene stool sample is less than the threshold value of the Ct value. Positive, the Ct value in the TLX2 gene stool specimen is greater than or equal to the cutoff value of the Ct value being negative.

In some specific embodiments of the present invention, the tumor of the present invention is a colorectal tumor.

In some specific embodiments of the present invention, the tumor of the present invention is colorectal cancer or adenoma.

In some specific embodiments of the present invention, the sample to be tested or the sample type provided by the present invention is tissue, body fluid or excrement.

In some embodiments of the invention, the tissue is intestinal tissue.

In some specific embodiments of the present invention, the body fluid includes blood, serum, plasma, extracellular fluid, interstitial fluid, lymph fluid, cerebrospinal fluid, or aqueous humor.

In some embodiments of the invention, the excreta is sputum, urine, saliva or feces.

Through research, the present invention finds that by jointly detecting the methylation level of the promoter region of the COL4A2 and / or TLX2 genes, colorectal cancer specimens can be distinguished from fecal specimens of normal people very accurately. The present invention detects colorectal cancer by using a methylation detection reagent containing these two genes, and the detection sensitivity and specificity for colorectal cancer are very high.

Compared with the existing markers for detecting bowel cancer, the markers and marker combinations provided by the present invention and the technical solution can detect colorectal cancer with extremely high sensitivity and specificity. The specific points are as follows:

1. In one of the above technical solutions, the methylation detection reagent containing the COL4A2 gene provided by the present invention can detect 92.5% of colorectal cancer when the specificity is 91.6%.

2. In one of the above technical solutions, the methylation detection reagent containing the TLX2 gene provided by the present invention can detect 88.8% of colorectal cancer when the specificity is 96.4%, which can be easily performed by stool. To detect samples, make a reliable diagnosis of colorectal cancer. Obtaining stool samples is very easy, sampling is non-invasive and simple, and it will not cause any pain and inconvenience to the patient.

3. In one verification, a methylation detection reagent containing a combination of COL4A2 and TLX2 genes was able to detect 91.3% of colorectal cancer in a stool specimen with a specificity of 98.8%. The combination reagent can very easily and accurately determine colorectal cancer and normal people. The methylation detection reagent of the gene is expected to be used in a stool gene detection kit and serve for clinical detection of colon cancer.

4. In one of the above technical solutions, whether the two genes COL4A2 and / or TLX2 are detected individually or in combination, they have extremely high sensitivity and specificity for colorectal cancer. Therefore, when other genes combine these two genes, When one or two genes are used to detect colorectal tumors, it is also possible to achieve high sensitivity and specificity similar to the present invention.

5. In one of the above technical solutions, the reagent / kit detects and diagnoses cancer by methylation level. More and more studies have confirmed that the methylation change is an early event in the process of tumorigenesis and detects abnormal methylation. It is easier to detect early lesions.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to explain the technical solutions in the embodiments of the present invention or the prior art more clearly, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

FIG. 1 shows a dot plot and a ROC curve of colorectal cancer detected by COL4A2 and TLX2 genes in a stool test of Example 1. FIG. 1 (A) shows a dot plot of colorectal cancer detected by COL4A2 and TLX2 genes in a stool test. Figure 1 (B) shows the ROC curve of colorectal cancer detected by COL4A2 and TLX2 genes;

Figure 2 shows the ROC curve of colorectal cancer detected by SFRP1 and CDH4 genes in 36 stool tests of the comparative example; of which, Figure 2 (A) shows the ROC curve of colorectal cancer detected by SFRP1 gene in 36 stool tests; Figure 2 (B) Shows the ROC curve of colorectal cancer detected by CDH4 gene in 36 stool tests.

Detailed ways

The invention discloses a tumor marker, a methylation detection reagent, a kit and an application thereof. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be noted that all similar replacements and modifications will be apparent to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can modify or appropriately modify and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention. Apply the technology of the present invention.

The tumor markers, methylation reagents, kits and the raw materials, auxiliary materials and reagents used in the application can be purchased or synthesized on the market.

Any nucleic acid fragment of the COL4A2 and / or TLX2 gene can be used in the present invention as long as there is a CpG site that can detect differential methylation. CpG islands are CpG-rich regions in a nucleic acid sequence. CpG islands start upstream of the promoter and extend downstream to the transcribed region. Methylation of CpG islands on the promoter usually suppresses gene expression. The CpG island in the promoter is part of the methylation, and there is a conserved DNA methylation target in the CpG open sea of the genome. Recent studies have revealed the synergistic effects of methylation of non-promoter regions (such as genomic and UTR) on gene expression. Genomic methylation may be a potential therapeutic target in cancer.

Generally speaking, CpG islands refer to some regions rich in CpG dinucleotides, which are usually located in the promoter and its vicinity. The CpG islands in the present invention not only refer to the promoter and its vicinity are rich in CpG dinucleus. Nucleotides also include hybrid methylated CpG sites, or isolated CpG sites.

Generally, the CpG-containing nucleic acid is DNA. However, the present invention is applicable, for example, a sample containing DNA, or DNA and RNA containing mRNA, wherein the DNA or RNA may be single-stranded or double-stranded, or a DNA-RNA hybrid strand may be included in the sample.

A "primer" or "probe" in the present invention refers to an oligonucleotide comprising a region complementary to the sequence of at least 6 consecutive nucleotides of a target nucleic acid molecule (eg, a target gene). In some embodiments, the primer or probe comprises 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 with the target molecule. A region of complementary sequence of consecutive or discontinuous block nucleotides. When a primer or probe comprises a region that is complementary to at least x consecutive nucleotides of the target molecule, the primer or probe is at least 95% complementary to at least x consecutive nucleotides of the target molecule. In some embodiments, the primer or probe is at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target molecule.

The "detection" in the present invention is the same as diagnosis. In addition to the early diagnosis of colorectal tumors, it also includes the diagnosis of middle and advanced stages of colorectal tumors. Selection of therapeutic targets.

The use of colorectal tumor markers COL4A2 and / or TLX2 genes makes early diagnosis of colorectal tumors possible. When a methylated gene is determined to be methylated in a clinically or morphologically normal cell in a cancer cell, this indicates that the normally expressed cell is developing towards cancer. In this way, colorectal cancer can be diagnosed at an early stage by methylation of the colorectal tumor-specific genes COL4A2 and / or TLX2 genes in normally-presented cells.

Among them, early diagnosis refers to the possibility of discovering 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 the present invention are also promising for colorectal tumor screening, risk assessment, prognostic diagnosis, disease identification, diagnosis of the disease stage, 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 COL4A2 and / or TLX2 genes obtained from a sample by the progression of colorectal tumors at different stages or stages. By comparing the degree of methylation of the COL4A2 and / or TLX2 gene genes of nucleic acids isolated from samples from each stage of colorectal cancer with one or more isolated from samples from intestinal tissues without abnormal cell proliferation The degree of methylation of the COL4A2 and / or TLX2 genes of nucleic acids can detect specific stages of colorectal tumors in a sample.

The following further describes the present invention in combination with the embodiments:

Example 1

Select 163 stool samples (80 colorectal cancer, 83 normal, all confirmed by colonoscopy or pathology), grind and centrifuge, add 100 μl capture magnetic beads (containing the capture sequence of COL4A2, TLX2 genes, and internal reference gene ACTB), And according to the technical scheme described below, finally 15 μl of DNA after Bisulfite transformation was obtained. Then qMSP was performed to detect the methylation levels of COL4A2 and TLX2. qMSP reaction system, reaction program and calculation method of methylation level are the same as those of the comparative example. A logistic regression model was used to construct a two-gene joint diagnosis model.

The technical scheme is as follows:

1) Collect stool samples of normal people and colorectal tumor patients with colonoscopy results, and mix and grind according to 1g of feces: 4mL protection solution, centrifuge at 5000rpm for 10min, remove the supernatant and discard the precipitate;

2) Take out 10mL of the supernatant and centrifuge again, take 3.2mL of the supernatant, add 2mL of lysate and 100μl capture magnetic beads M1, incubate at 92 ℃ for 10min, and then leave it at room temperature for 1h;

3) Discard the supernatant on a magnetic stand, wash the magnetic beads and transfer to a 2 mL centrifuge tube, add 800 μl of washing solution W1, and incubate for 1 min at 1300 rpm at room temperature. Aspirate the supernatant and repeat 3 times

4) Add 55 μl of eluate, incubate at 1300 rpm at 92 ° C for 10 minutes, place on a magnetic stand, and transfer 50 μl of eluate to a new EP tube within 3 minutes;

5) EZ DNA DNA Kit (Zymo Research) was used to methylate the DNA fragment in the previous step, and the final eluent was 15ul for qMSP detection.

qMSP reaction system: 25ul (nuclease-free water 8.2μl, 5 × Colorless GoTaq Flexi buffer 5μl, MgCl2 (25mM) 5μl, dNTPs (10mM) 1μl, GoTaq Hot Start Polymerase 0.5μl, Forward Primer (100μM) 0.125μl, Reverse Primer (100 μM) 0.125 μl, Probe (100 μM) 0.05 μl, DNA 5 μl). Reaction procedure: 95 ° C for 4 min, (95 ° C for 20s, 56 ° C for 30s, 72 ° C for 30s) × 45 Cycles, 37 ° C for 30s.

Finally, the copy number of the gene in the specimen was calculated according to the standard curve (the method refers to the previous research results: Niu F, Wen J, Fu X, Li C, Zhao R, Wu S, Yu H, Liu X, Zhao X, Liu S, Wang X, Wang J, Zou H. Stool DNA test of MethylatedSyndecan-2 for the early detection of colorectal neoplasia.CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION 2017; 26 (9): 1411-1419.).

The methylation sites of COL4A2 and TLX2 genes are relatively constant, and they are mainly located in the promoter region or nearby CpG islands. A set of preferred capture sequences, primers and probes were designed for these regions and used in COL4A2, TLX2 gene methylation detection reagents.

The capture sequences and primer probes contained in the reagent are as follows:

Capture sequence of COL4A2 (SEQ ID NO: 1):

5’-GCTGCTGCCCGAACGCATTGGCCCTTCCAGAAGCA-3 ’

QMSP primer probe for COL4A2:

Forward Primer (SEQ ID NO: 2): 5’-AGAGAGTTTAGTAAGGTCGGTC-3 ’

Reverse Primer (SEQ ID NO: 3): 5’-GACTTCAAAAACTACTACCCG-3 ’

Probe (SEQ ID NO: 4): 5’-TGTCGGTGTGTCGTCGGC-3 ’

Capture sequence of TLX2 (SEQ ID NO: 5):

5’-CCAGCGGGCGGCGGCACAGGCAGCGGGCGGTGCGCAGGGA-3 ’

QMSP primer probes for TLX2:

Forward Primer (SEQ ID NO: 6): 5’-ATTACGGAGTTTCGGGTTAC-3 ’

Reverse Primer (SEQ ID NO: 7): 5’-GACGACACAAACAACGAACG-3 ’

Probe (SEQ ID NO: 8): 5’-CGTTGTTCGGTAGTTTCGGAGTGG-3 ’

In the stool test, the dot plot and ROC curve of colorectal cancer detected by COL4A2 and TLX2 genes are shown in Figure 1 (A) and Figure 1 (B):

For colorectal cancer, the detection sensitivity of the COL4A2 gene was 92.5%, the specificity was 91.6%, and the area under the ROC curve was 0.966 (95% CI: 0.941-0.991, p <0.0001). The detection sensitivity of TLX2 gene was 88.8%, the specificity was 96.4%, and the area under the ROC curve was 0.955 (95% CI: 0.922-0.989, p <0.0001). When the two genes are combined, the specificity is 98.8% (82/83), the sensitivity is 91.3% (73/80), and the area under the ROC curve is 0.985 (95% CI: 0.971-1, p <0.0001). ). The results show that in the case of the combined diagnosis of the two genes, the normal population and colorectal cancer patients can be distinguished with very high sensitivity and specificity.

Table 1

Table 1 Note: "No amplification" means no amplification curve and no Ct data, which belongs to a range greater than the cutoff.

Comparative Example 1

Studies have shown that methylation of the SFRP1 gene is related to bowel cancer, and detecting the degree of methylation of this gene in stool can detect colorectal cancer. In 53 fecal specimens (29 cases of bowel cancer, 7 cases of adenoma, and 17 cases of normal), 89% of colorectal tumors could be detected with a specificity of 86%. (Zhang, W, Bauer, M, Croner, RS, Pelz, JO, Lodygin, D, Hermeking, H, Sturzl, M, Hohenberger, W, Matzel, KE.DNA, tool test for colorectal, cancer: Hypermethylation, of the secreted, frizzled-related, and less COLON & RECTUM 2007; 50 (10): 1618-26; discussion 1626-7.). Other studies have shown that the methylated CDH4 gene can also be used as a marker for detecting colorectal cancer. The methylated CDH4 gene can detect 78% (38/49) of colorectal cancer, with a specificity of 100% (0/10) (Frequent Aberrant Methylation of the CDH4 Gene Promoter in Human Colorectal and Gastric Cancer.CANCER RESEARCH. 2004)

Comparative Example 2

The methylation levels of SFRP1 and CDH4 genes were also detected in 36 stool samples. Select 36 fecal specimens (18 colorectal cancer, 18 normal, all confirmed by colonoscopy or pathology), grind and centrifuge, and add 100 μl capture magnetic beads (capture sequences containing SFRP1 and CDH4 genes, and internal reference gene ACTB), According to the following technical scheme, 15ul of DNA converted from Bisulfite was finally obtained. Then qMSP was performed to detect the methylation levels of SFRP1 and CDH4.

The technical scheme is as follows:

1) Collect stool samples of normal people and colorectal tumor patients with colonoscopy results, and mix and grind according to 1g of feces: 4mL protection solution, centrifuge at 5000rpm for 10min, remove the supernatant and discard the precipitate;

2) Take out 10mL of the supernatant and centrifuge again, take 3.2mL of the supernatant, add 2mL of lysate and 100μl capture magnetic beads M1, incubate at 92 ℃ for 10min, and then leave it at room temperature for 1h;

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

4) Add 55 μl of eluate, incubate at 1300 rpm at 92 ° C for 10 minutes, place on a magnetic stand, and transfer 50 μl of eluate to a new EP tube within 3 minutes;

5) EZ DNA DNA Kit (Zymo Research) was used to methylate the DNA fragment in the previous step, and the final eluate was 15 μl for qMSP detection.

qMSP reaction system: 25 μl (8.2 μl of nuclease-free water, 5 μl of Colorless GoTaq Flexi Buffer 5 μl, 5 μl of MgCl ₂ (25 mM), 1 μl of dNTPs (10 mM), 0.5 μl of GoTaq Hot Start polymerase, 0.125 μl of Forward primer (100 uM), Reverse primer (100 μM) 0.125 μl, Probe (100 uM) 0.05 μl, DNA 5 μl). Reaction procedure: 95 ° C for 4 min, (95 ° C for 20s, 56 ° C for 30s, 72 ° C for 30s) × 45 Cycles, 37 ° C for 30s.

Finally, the copy number of the gene in the specimen was calculated according to the standard curve (the method refers to the previous research results: Niu F, Wen J, Fu X, Li C, Zhao R, Wu S, Yu H, Liu X, Zhao X, Liu S, Wang X, Wang J, Zou H. Stool DNA test of MethylatedSyndecan-2 for the early detection of colorectal neoplasia.CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION 2017; 26 (9): 1411-1419.).

The ROC curves of colorectal cancer detected by SFRP1 and CDH4 genes in 36 stool tests are shown in Figure 2 (A) and Figure 2 (B):

For colorectal cancer, the detection sensitivity of the SFRP1 gene was 67%, the specificity was 94%, and the area under the ROC curve was 0.892 (95% CI: 0.790-0.994, p <0.0001). The detection sensitivity of CDH4 gene is also 67%, the specificity is 94%, and the area under the ROC curve is 0.867 (95% CI: 0.745-0.990, p <0.001). When the two genes are combined, the test interpretation criteria are: when at least one gene is positive, the interpretation result is a tumor specimen; when both genes are negative, the interpretation result is a normal specimen. Tumor specimens and normal specimens are judged based on the Ct threshold value of the predicted probability when the two genes are jointly diagnosed. The Ct threshold value in the SFRP1 gene stool sample is 32, which is less than the threshold value positive and greater than or equal to the threshold value; in the CDH4 gene stool sample. The Ct cutoff is 33, the cutoff is positive, and the cutoff is negative. When the two genes are combined, the sensitivity is 67% (12/18) with a specificity of 94% (17/18).

Table 2

Table 2 Note: "No amplification" means no amplification curve and no Ct data, which belongs to a range greater than the cutoff value.

From the above data as a comparison, it can be seen that the detection of TLX2 gene, the combined detection of COL4A2 gene and TLX2 gene have higher sensitivity and specificity for colorectal cancer, and the effect is better.

The above is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and retouches can be made. These improvements and retouches also It should be regarded as the protection scope of the present invention.

Claims (14)

Application of TLX2 gene and / or COL4A2 gene in preparing tumor markers; Preferably, the sequence of the TLX2 gene has at least 97.8%, at least 98.9%, at least 99.9% or 100% identity with the sequence shown in Genebank Accession No. NC_000002.12; Preferably, the sequence of the COL4A2 gene has at least 97.8%, at least 98.9%, at least 99.9% or 100% identity with the sequence shown in Genebank Accession No. NC_000013.11; Preferably, the tumor is a colorectal tumor; Preferably, the tumor is colorectal cancer or adenoma; Preferably, the sample to be tested for the marker is tissue, body fluid or excreta; Preferably, the tissue is intestinal tissue; Preferably, the body fluid is blood, serum, plasma, extracellular fluid, interstitial fluid, lymph fluid, cerebrospinal fluid or aqueous humor; Preferably, the excreta is sputum, saliva, urine or feces. Application of methylation detection reagent of TLX2 gene and / or COL4A2 gene in preparing tumor detection reagent or kit; Preferably, the sequence of the TLX2 gene has at least 97.8%, at least 98.9%, at least 99.9% or 100% identity with the sequence shown in Genebank Accession No. NC_000002.12; Preferably, the sequence of the COL4A2 gene has at least 97.8%, at least 98.9%, at least 99.9% or 100% identity with the sequence shown in Genebank Accession No. NC_000013.11; Preferably, the tumor is a colorectal tumor; Preferably, the tumor is colorectal cancer or adenoma; Preferably, the test sample targeted by the detection reagent is tissue, body fluid or excreta; Preferably, the tissue is intestinal tissue; Preferably, the body fluid is blood, serum, plasma, extracellular fluid, interstitial fluid, lymph fluid, cerebrospinal fluid or aqueous humor; Preferably, the excreta is sputum, urine, saliva or feces. A capture sequence, wherein the capture sequence has any one of the nucleotide sequences shown below: XVII. It has the nucleotide sequence shown in SEQ ID NO: 5; XVIII. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 5 or a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5 Functionally similar nucleotide sequences obtained from CpG islands; XIX. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 5 or having the core shown in SEQ ID NO: 1 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; XX, the complement of a sequence shown as XVII, XVIII or XIX. A primer pair characterized in that the upstream primer has any one of the nucleotide sequences shown below: XXI, having the nucleotide sequence shown in SEQ ID NO: 6; XXII. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 6; XXIII. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 6 or having the core shown in SEQ ID NO: 6 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; XXIV, the complement of a sequence shown by XXI, XXII or XXIII; The downstream primer has any of the nucleotide sequences shown below: XXV, having the nucleotide sequence shown in SEQ ID NO: 7; XXVI, a nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 7; XXVII. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 7 or having a core shown in SEQ ID NO: 7 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; XXVIII, the complement of a sequence shown by XXV, XXVI or XXVII. A probe characterized in that the probe has any one of the nucleotide sequences shown below: XXIX, having the nucleotide sequence shown in SEQ ID NO: 8; XXX. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 8; XXXI, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 8 or having the core shown in SEQ ID NO: 8 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; XXXII, the complement of the sequence shown by XXIX, XXX or XXXI. COL4A2 gene and / or TLX2 gene methylation detection reagent, characterized in that it includes capture sequences, primers and / or probes for COL4A2 gene and / or TLX2 gene; Preferably, it comprises capture sequences, primers and / or probes obtained against CpG islands of the COL4A2 gene and / or the TLX2 gene; Preferably, it comprises capture sequences, primers, and / or probes obtained for genomic, intergenic, promoter, or CpG islands in the vicinity of the promoter region of the COL4A2 gene and / or TLX2 gene; Preferably, the capture sequence of the COL4A2 gene has any one of the nucleotide sequences shown below: I. It has the nucleotide sequence shown in SEQ ID NO: 1; II. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 1 Functionally similar nucleotide sequences obtained from CpG islands; III. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 1 or having a core shown in SEQ ID NO: 1 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; IV. The complement of the sequence shown in I, II or III; Preferably, the upstream primer of the COL4A2 gene has any one of the nucleotide sequences shown below: V, having the nucleotide sequence shown in SEQ ID NO: 2; VI. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 2; VII. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 2 or having a core shown in SEQ ID NO: 2 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; VIII, the complement of the sequence shown in V, VI or VII; The downstream primer of the COL4A2 gene has any one of the nucleotide sequences shown below: IX. It has the nucleotide sequence shown in SEQ ID NO: 3; X. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 3; XI. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 3 or having the core shown in SEQ ID NO: 3 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; XII, the complement of the sequence shown by IX, X or XI; Preferably, the probe of the COL4A2 gene has any one of the nucleotide sequences shown below: XIII, having the nucleotide sequence shown in SEQ ID NO: 4; XIV. A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 4; XV, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 4 or having the core shown in SEQ ID NO: 4 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; XVI, the complement of a sequence such as XIII, XIV or XV; Preferably, the capture sequence of the TLX2 gene has any one of the nucleotide sequences shown below: XVII. It has the nucleotide sequence shown in SEQ ID NO: 5; XVIII. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 5 or a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 5 Functionally similar nucleotide sequences obtained from CpG islands; XIX. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 5 or having the core shown in SEQ ID NO: 1 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; XX, the complement of a sequence shown by XVII, XVIII or XIX; Preferably, the upstream primer of the TLX2 gene has any one of the nucleotide sequences shown below: XXI, having the nucleotide sequence shown in SEQ ID NO: 6; XXII. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 6; XXIII. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 6 or having the core shown in SEQ ID NO: 6 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; XXIV, the complement of a sequence shown by XXI, XXII or XXIII; The downstream primer of the TLX2 gene has any one of the nucleotide sequences shown below: XXV, having the nucleotide sequence shown in SEQ ID NO: 7; XXVI, a nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 7; XXVII. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 7 or having a core shown in SEQ ID NO: 7 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; XXVIII, the complement of the sequence shown by XXV, XXVI or XXVII; Preferably, the probe of the TLX2 gene has any one of the nucleotide sequences shown below: XXIX, having the nucleotide sequence shown in SEQ ID NO: 8; XXX. A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 8; XXXI, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 8 or having the core shown in SEQ ID NO: 8 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences; XXXII, the complement of the sequence shown by XXIX, XXX or XXXI. A kit comprising the capture sequence according to claim 3, or the primer pair according to claim 4, or the probe according to claim 5, or the methyl group according to claim 6. Chemical detection reagent Preferably, the kit includes: a first container containing a capture sequence; a second container containing a primer pair for amplification; and a third container containing a probe. The capture sequence according to claim 3, or the primer pair according to claim 4, or the probe according to claim 5, or the methylation detection reagent according to claim 6, and a kit for detecting tumor Application Preferably, the tumor is a colorectal tumor; Preferably, the tumor is colorectal cancer or adenoma; Preferably, the sample to be tested is tissue, body fluid or excreta; Preferably, the tissue is intestinal tissue; Preferably, the body fluid is blood, serum, plasma, extracellular fluid, interstitial fluid, lymph fluid, cerebrospinal fluid or aqueous humor; Preferably, the excreta is sputum, saliva, urine or feces. The capture sequence according to claim 3, or the primer pair according to claim 4, or the probe according to claim 5, or the methylation detection reagent according to claim 6, or the reagent according to claim 7. Application of the box in detecting tumors; Preferably, the tumor is a colorectal tumor; Preferably, the tumor is colorectal cancer or adenoma; Preferably, the sample to be tested is tissue, body fluid or excreta; Preferably, the tissue is intestinal tissue; Preferably, the body fluid is blood, serum, plasma, extracellular fluid, interstitial fluid, lymph fluid, cerebrospinal fluid or aqueous humor; Preferably, the excreta is sputum, urine, saliva or feces. A tumor detection method, characterized in that the method includes: (1) detecting the methylation level of COL4A2 gene and / or TLX2 gene in a subject; (2) comparing the methylation level of the subject's COL4A2 gene and / or TLX2 gene with that of a normal control sample; (3) According to an increase in the methylation level of the TCOL4A2 gene and / or TLX2 gene of the subject compared to the methylation level of a normal control sample, it indicates that the subject has or is at risk of developing a tumor To distinguish between normal and tumor samples; Preferably, by detecting the methylation level of the COL4A2 gene and / or the TLX2 gene genome, intergenic region or promoter region and the region near the promoter region; Preferably, a normal sample and a tumor sample are distinguished by detecting methylation levels in the promoter region of the COL4A2 gene and / or the TLX2 gene and a region near the promoter region; Preferably, the methylation level is through methylation-specific PCR, or methylation-specific quantitative PCR, or methylation-specific DNA-binding protein PCR, quantitative PCR, and DNA chip, or methylation-sensitive Restriction enzymes, or bisulfite sequencing, or pyrosequencing; Preferably, the methylation level is detected by methylation-specific quantitative PCR; Preferably, the methylation level uses a capture sequence according to claim 3, or a primer pair according to claim 4, or a probe according to claim 5, or a methylation detection according to claim 6. Reagent, or kit detection according to claim 7; Preferably, in step (1), detecting the methylation level of the subject's COL4A2 gene and / or TLX2 gene comprises the following steps: a) Use the magnetic bead capture method to extract the DNA of the sample to be tested; b) the DNA of the test sample is transformed with bisulfite, bisulfite or hydrazine; c) methylation-specific quantitative PCR detection; Preferably, in step a), extracting the DNA of the sample to be tested by using a magnetic bead capture method includes the following steps; Take the sample to be tested, mix and grind it in the protection solution, centrifuge, and take the supernatant; Supernatant was centrifuged, the supernatant was taken, lysate and magnetic beads with specific complementary oligonucleotide capture sequences were added to the supernatant and incubated; After discarding the supernatant, the magnetic beads were washed and transferred to a clean centrifuge tube, and the washing solution was added, and incubated at room temperature at 100-2000 rpm for 0.5-5min. The supernatant was placed on a magnetic stand and the supernatant was repeated 3 times; The target gene DNA was eluted with a buffer. The method according to claim 10, wherein the detection standard is: COL4A2 gene and / or TLX2 gene combined detection, and when at least one gene test result is positive, the interpretation result is a tumor specimen; When the test results of all genes are negative, the interpretation result is a normal specimen; The test result is judged to be positive or negative according to the cutoff value of the Ct value of the two genes. The cutoff value of the Ct value in the stool sample of the COL4A2 gene is 30 to 40, and the cutoff value of the Ct value in the stool sample of the COL4A2 gene is less than the cutoff value of the Ct value. Positive, the Ct value in the COL4A2 stool sample is greater than or equal to the cutoff value of the Ct value; The cutoff value of the Ct value in the TLX2 gene stool sample is 32 to 42, the Ct value in the TLX2 gene stool sample is less than the cutoff value of the Ct value is positive, and the Ct value in the TLX2 gene stool sample is greater than or equal to the Ct value. The cutoff is negative. A tumor detection system, characterized in that the system includes the following components; (1) The methylation detection component of the COL4A2 gene and / or the TLX2 gene; (2) data processing components; (3) result output component; Preferably, the methylation detection component contains one or more of a fluorescent quantitative PCR instrument, a PCR instrument, and a sequencer; Preferably, the methylation detection member further comprises a capture sequence according to claim 3, or a primer pair according to claim 3, or a probe according to claim 3, or according to claim 6. Methylation detection reagent, or the kit according to claim 7; Preferably, the data processing component 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 of test sample Test data of samples and normal control samples; d. According to the comparison results, in response to the probability or possibility of the test subject developing a tumor; Preferably, the result output component is used to output a probability or possibility that the test subject has a tumor; Preferably, the judgment criterion of the data processing component is: judge the tumor specimen and the normal specimen according to the cutoff value; The COL4A2 gene and / or TLX2 gene are jointly tested. When at least one gene is positive, the interpretation result is a tumor specimen; when both genes are negative, the interpretation result is a normal specimen; The test result is judged to be positive or negative according to the cutoff value of the Ct value of the two genes. The cutoff value of the Ct value in the stool sample of the COL4A2 gene is 30 to 40, and the cutoff value of the Ct value in the stool sample of the COL4A2 gene is less than the cutoff value of the Ct value. Positive, the Ct value in the COL4A2 stool sample is greater than or equal to the cutoff value of the Ct value; The cutoff value of the Ct value in the TLX2 gene stool sample is 32 to 42, the Ct value in the TLX2 gene stool sample is less than the cutoff value of the Ct value is positive, and the Ct value in the TLX2 gene stool sample is greater than or equal to the Ct value. The cutoff is yin. The method according to any one of claims 10-11, or the system according to claim 12, wherein the tumor is a colorectal tumor; Preferably, the tumor is colorectal cancer or adenoma. The method according to any one of claims 10-11, or the system according to claim 12, wherein the system or method detects a sample type as tissue, body fluid or excreta; Preferably, the tissue is intestinal tissue; Preferably, the body fluid is blood, serum, plasma, extracellular fluid, interstitial fluid, lymph fluid, cerebrospinal fluid or aqueous humor; Preferably, the excreta is sputum, urine, saliva or feces.

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