WO2019223519A1 - 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 TMEM240 gene promoter region. The present invention detects colorectal cancer using a methylation detection reagent containing the gene.

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.

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; 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 is used in tissues. The sensitivity was 83%, which was reduced to 46% in fecal specimens. Similar SFRP1, SFRP2 and other genes were also found. Such markers could not meet the needs for clinical detection of colon cancer. Therefore, the selection 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. This tumor marker is very sensitive to bowel 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 TMEM240 gene in preparing tumor markers.

In some specific embodiments of the present invention, the sequence of the TMEM240 gene is at least 97.8% identical to the sequence shown in Genebank Accession No. NC_000001.11.

In some specific embodiments of the present invention, the sequence of the TMEM240 gene is at least 98.9% identical to the sequence shown in Genebank Accession No. NC_000001.11.

In some specific embodiments of the present invention, the sequence of the TMEM240 gene is at least 99.9% identical to the sequence shown in Genebank Accession No. NC_000001.11.

In some specific embodiments of the present invention, the sequence of the TMEM240 gene is 100% identical to the sequence shown in Genebank Accession No. NC_000001.11.

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 present invention, the body fluid includes, but is not limited to, 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.

The invention also provides the application of the methylation detection reagent of the TMEM240 gene in preparing a tumor detection reagent or a kit.

The methylation detection reagent of the TMEM240 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, bisulfite sequencing, or Pyrosequencing and more. Each detection method has its corresponding reagent, and these reagents can be used to detect methylation of the TMEM240 gene using the present invention.

The invention also provides a capture sequence, which 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.

The present invention also provides a primer pair, and the upstream primer 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;

The downstream primer has any 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.

The present invention also provides a probe having 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.

The present invention also provides a methylation detection reagent for the TMEM240 gene, including capture sequences, primers, and / or probes for the TMEM240 gene.

In some specific embodiments of the present invention, capture sequences, primers, and / or probes obtained for the CpG island of the TMEM240 gene are included.

In some specific embodiments of the present invention, the primers and / or probes detect methylation of the TMEM240 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 the methylation level of the genomic body, the intergenic region or the promoter region of the TMEM240 gene and a region near the promoter region.

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 provided by the present invention includes capture sequences, primers, and / or probes obtained for a promoter region of the TMEM240 gene or a CpG island in a region near the promoter region.

In some specific embodiments of the present invention, the capture sequence in the methylation detection reagent provided by the present invention 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, in the methylation detection reagent provided by the present invention, the upstream primer in the primer 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;

VII, the complement of the sequence shown in V, VI or VII.

In some specific embodiments of the present invention, in the methylation detection reagent provided by the present invention, the downstream primer in the primer 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 in the methylation detection reagent provided by the present invention 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.

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, and 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 polymerase, dNTPs, Mg ²⁺ ions and buffers commonly used in the amplification of TMEM240 gene.

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 TMEM240 gene.

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

(1) detecting the methylation level of the subject's TMEM240 gene;

(2) comparing the methylation level of the subject's TMEM240 gene with that of a normal control sample;

(3) According to the increase in the methylation level of the TMEM240 gene of the subject compared to the methylation level of the normal control sample, indicating that the subject has or is at risk of developing a tumor to distinguish normal samples And tumor samples.

In some specific embodiments of the present invention, the present invention detects the methylation level of the genomic body, the intergenic region or the promoter region of the TMEM240 gene and a region near the promoter region.

In some specific embodiments of the present invention, the present invention distinguishes normal samples from tumor samples by detecting the methylation level of the promoter region of the TMEM240 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 TMEM240 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.

In some specific embodiments of the present invention, in step a), extracting DNA from a 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.

In some specific embodiments of the present invention, the detection standard is: judging tumor specimens and normal specimens according to cutoff values, the cutoff value of Ct values in fecal specimens is 32 to 42, preferably, cutoff values of Ct value in feces specimens Is 35, the Ct value of the fecal specimen is less than the cutoff value of the Ct value is judged as a tumor specimen, and the Ct value of the feces specimen is greater than or equal to the cutoff value of the Ct value as a normal specimen. 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) TMEM240 gene methylation detection component;

(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 criterion of the data processing component is:

The tumor specimen and the normal specimen are judged according to the cutoff value. The cutoff value of the Ct value in the stool specimen is 32 to 42, preferably, the cutoff value of the Ct value in the stool specimen is 35, and the Ct value of the stool specimen is less than the Ct. The cutoff value of the value is judged as a tumor specimen, and the cutoff value of the Ct value of the fecal specimen is greater than or equal to the cutoff value of the Ct value as a normal specimen. This threshold can be adjusted according to the actual situation.

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.

The present invention finds through research that by detecting the methylation level of the promoter region of the TMEM240 gene, colorectal cancer specimens can be distinguished well from normal human stool specimens. The present invention detects colorectal cancer by using a methylation detection reagent containing the gene, and the detection sensitivity and specificity for colorectal cancer are very high. The present invention has for the first time verified that there is a correlation between the methylation level of this gene and the onset of colorectal cancer.

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

1. In one of the above technical solutions, the methylation detection reagent containing the TMEM240 gene provided by the present invention can detect 83% of colorectal cancer in a stool sample with a specificity of 94%, 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.

2. In one of the above technical solutions, the methylation detection reagent and extraction detection method containing the TMEM240 gene can very easily and accurately determine colorectal cancer and normal people. The methylation detection reagent of this gene is expected to be used in feces. Genetic test kits and serve for clinical detection of bowel cancer.

3. The reagent / kit in one of the above technical solutions is to detect and diagnose cancer by methylation level. More and more studies have confirmed that the methylation change is an early event in 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.

Figure 1 shows the ROC curve of colorectal cancer detected by TMEM240 gene in the stool test of Example 1;

FIG. 2 shows the amplification curve of the TMEM240 gene standard curve in the stool experiment of Example 1. FIG.

Figure 3 shows the ROC curve of colorectal cancer detected by the SFRP1 gene in a 19-pair tissue experiment of Comparative Example 2;

Figure 4 shows the ROC curve of colorectal cancer detected by the SFRP1 gene in the 36 stool tests of Comparative Example 2.

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 by the preferred embodiments. Obviously, relevant persons can make modifications or appropriate changes and combinations to 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.

As long as there is a CpG site that can detect differential methylation, any nucleic acid fragment of the TMEM240 gene can be used in the present invention. 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, and also includes colorectal tumor screening, risk assessment, prognosis, disease identification, diagnosis of disease stages, and Selection of therapeutic targets.

The application of colorectal tumor marker TMEM240 makes 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 normally expressed cell is developing toward cancer. In this way, colorectal cancer can be diagnosed at an early stage by methylation of the colorectal tumor-specific gene TMEM240 in normal-representing 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 progress of colorectal tumors at different stages or stages by measuring the degree of methylation of TMEM240 obtained from a sample. By comparing the degree of methylation of TMEM240 genes of nucleic acids isolated from samples from each stage of colorectal cancer with TMEM240 genes of one or more nucleic acids isolated from samples from intestinal tissues without abnormal cell proliferation The degree of basicization can detect the specific stage of colorectal tumor in the sample.

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

Example 1

Select 36 fecal specimens (18 colorectal cancer, 18 normal, all confirmed by colonoscopy or pathology), grind and centrifuge, add 100ul capture magnetic beads (capture sequence containing TMEM240 gene), and follow the technical scheme described above Operation, and finally got 15ul of DNA transformed with Bisulfite. Then qMSP was performed to detect the methylation level of TMEM240.

qMSP reaction system: 25ul (nuclease-free water 8.2ul, 5 × Colorless GoTaq FlexiBuffer 5ul, MgCl2 (25mM) 5ul, dNTPs (10mM) 1ul, GoTaq Hot Start Polymerase 0.5ul, Forward primer (100uM) 0.125ul, Reverse Primer (100uM) 0.125ul, Probe (100uM) 0.05ul, DNA 5ul). 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.

TMEM240 gene methylation site is relatively constant, mainly located in the promoter region or nearby CpG island. A set of capture sequences, primers and probes were designed for these regions and used in the TMEM240 gene methylation detection reagent.

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

Capture sequence of TMEM240 (SEQ ID NO: 1):

5’-GGTTGTGGACTCACCGGCCACAGTTGCAGTGGCAGACGC-3 ’

QMEMP primer probe for TMEM240:

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

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

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

The ROC curve of colorectal cancer detected by TMEM240 gene in feces experiment is shown in Figure 1:

For colorectal cancer, the detection sensitivity of TMEM240 gene was 83% (15/18), specificity was 94% (17/18), and the area under the ROC curve was 0.949 (95% CI: 0.883-1, p <0.0001)

In the stool experiment, the TMEM240 gene standard curve amplification map is shown in Figure 2:

The standard amplification efficiency was 101%, and the linearity R ^{2 was} 0.999.

Table 1 Raw data for stool experiments

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

At present, QIAamp DNA Stool Mini Kit (QIAGEN) is used to extract the DNA from stool samples, and then use methylation-specific PCR (MSP) or quantitative methylation-specific PCR (qMSP) to qualitatively or quantitatively detect the markers in the samples. Level. Among them, the detection of colorectal cancer by MSP requires running electrophoresis, which is more inconvenient and has the risk of product contamination. The QIAamp DNA Stool Mini Kit is used to extract fecal DNA from human and bacterial total DNA. There is very little human tumor DNA. , Is not conducive to subsequent PCR detection.

Comparative Example 2

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.)

The methylation level of SFRP1 gene was also detected in 19 pairs of tissues and 36 stool samples. The target gene extraction method in the stool samples was the same as in Example 1. In the 19 pairs of tissue experiments, QIAamp DNA Kit (QIAGEN) was used according to Protocol. Tissue DNA was extracted and transformed with EZ DNA DNA Kit (Zymo Research).

Then qMSP was performed to detect the methylation level of SFRP1.

The qMSP reaction system and reaction steps were the same as those in the stool test of Example 1. Finally, calculate the methylation value of the gene in the specimen according to the standard curve: (Target / ACTB) * 100. The qMSP primer probe used was the same as in Example 1.

The ROC curve of colorectal cancer detected by SFRP1 gene is shown in Figure 4:

For colorectal cancer tissue, the detection sensitivity of SFRP1 gene was 89%, the specificity was 95%, and the area under the ROC curve was 0.972 (95% CI: 0.929-1, p <0.001).

The ROC curve of colorectal cancer detected by SFRP1 gene in 36 stool tests is shown in Figure 5:

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).

It can be seen that the SFRP1 gene has a higher detection sensitivity and specificity for colorectal cancer tissues, and its sensitivity has been greatly reduced in stool samples.

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 principle of the present invention, a number of 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)

1. Application of TMEM240 gene in the preparation of tumor markers;

   Preferably, the sequence of the TMEM240 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_000001.11;

   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.
2. Application of methylation detection reagent of TMEM240 gene in preparing tumor detection reagent or kit;

   Preferably, the sequence of the TMEM240 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_000001.11;

   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.
3. A capture sequence, wherein the capture sequence 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. Complementary sequence to the sequence shown in I, II or III.
4. A primer pair characterized in that the upstream primer 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 has any 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 a sequence such as IX, X or XI.
5. A probe characterized in that the probe 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.
6. TMEM240 gene methylation detection reagent, characterized in that it includes capture sequences, primers and / or probes for methylation detection of TMEM240 gene;

   Preferably, it comprises a capture sequence, primers and / or probes obtained for the CpG island of the TMEM240 gene;

   Preferably, it comprises capture sequences, primers and / or probes obtained for the genomic body, intergenic region, promoter region of the TMEM240 gene, or CpG islands in the vicinity of the promoter region;

   Preferably, the capture sequence 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 in the primer 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 in the primer 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 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.
7. 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.
8. 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 a tumor is prepared. Application

   Preferably, the tumor is a bowel cancer;

   Preferably, the tumor is colorectal cancer or adenoma;

   Preferably, the sample to be tested is a body fluid or an excrement;

   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.
9. 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.
10. A tumor detection method, characterized in that the method includes:

    (1) detecting the methylation level of the subject's TMEM240 gene;

    (2) comparing the methylation level of the subject's TMEM240 gene with that of a normal control sample;

    (3) According to the increase in the methylation level of the TMEM240 gene of the subject compared to the methylation level of the normal control sample, indicating that the subject has or is at risk of developing a tumor to distinguish normal samples And tumor samples;

    Preferably, by detecting the methylation level of the genomic body, the intergenic region or the promoter region of the TMEM240 gene and a 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 TMEM240 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 nail according to claim 6. Basic detection reagent, or kit detection according to claim 7;

    Preferably, in step (1), detecting the methylation level of the subject's TMEM240 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.
11. The method according to claim 10, wherein the detection standard is: judging a tumor specimen and a normal specimen according to a cutoff value, a cutoff value of the Ct value in the stool specimen is 32 to 42, and the Ct value of the stool specimen is less than that The cutoff value of the Ct value is judged as a tumor specimen, and the cutoff value of the Ct value of the fecal specimen is greater than or equal to the cutoff value of the Ct value as a normal specimen.
12. A tumor detection system, characterized in that the system includes the following components;

    (1) TMEM240 gene methylation detection component;

    (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 4, or a probe according to claim 5, 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 boundary value of the Ct value in the stool sample is 32 to 42. The Ct value of the stool sample is less than the limit value of the Ct value, and it is determined to be a tumor sample. The value is judged as a normal specimen.
13. 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.
14. 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.

Images