Application of CGI methylation phenotype genome in preparing a kit for colorectal cancer typing or prognosis

Abstract

The present invention relates to the technical field of medical biological detection. The present invention provides a new use of the CGI methylation phenotype genome, specifically its application in the preparation of a kit for colorectal cancer CIMP typing or prognostic diagnosis. The CGI methylation phenotype genome is a combination of five genes, namely LRIF1, FOXL2, KIT, EPB41L4A, and B3GALT1. The present invention further provides a kit and a detection method for colorectal cancer CIMP typing diagnosis and prognostic evaluation by using quantitative PCR detection targeting the CGI methylation phenotype genome. The kit and the detection method of the present invention are simple, reliable, short in cycle, high in specificity, and easy to promote clinically.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biological detection, and relates to marker genes for CpG island (CGI) methylation phenotype (CIMP) typing and their application in the typing diagnosis and prognosis evaluation of colorectal cancer. Background Art

[0002] Colorectal cancer refers to cancers originating from the large intestine epithelium, including colon cancer and rectal cancer. The pathological type is most commonly adenocarcinoma, and very few are squamous cell carcinomas. Worldwide, colorectal cancer is the third most common malignant tumor and the second most common cause of malignant tumor death. In China, rectal cancer is the most common, followed by colon cancer.

[0003] Genotyping diagnosis and treatment is one of the research hotspots in the current treatment of colorectal cancer. In 1999, Minoru Toyota et al. found that abnormal methylation of CGI (CGI) in the promoter region was related to the transcriptional inactivation of tumor suppressor genes, and thus proposed the concept of colorectal cancer CGI methylation phenotype (CpG Island Methylation Phenotype, CIMP). Research has pointed out that colorectal cancers with different CIMP states have different clinical, pathological and molecular characteristics. For example, CIMP(+) colorectal cancers are more common in the proximal colon and women, with a high proportion of poorly differentiated tumors, microsatellite instability (MSI), a high BRAF mutation rate and a low p53 mutation rate, etc. Promoter CGI methylation is involved in the early carcinogenesis process of colorectal cancer.

[0004] However, there is no consensus on what methylation sites are used to judge the CIMP state, methylation detection methods and the definition of cut-off values, and there are some cases with opposite conclusions in different laboratories. To solve this problem, Weisenberger et al. used methylation fluorescence technology to screen out 5 CIMP marker genes (classical CIMP sites), namely CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1, from 195 CGIs. Subsequently, these 5 sites have been adopted in a large number of studies. However, some studies have pointed out that the classical CIMP sites lack specificity. For example, IGF2 and NEUROG1 also show high methylation in non-CIMP tumors. In addition, Herbst A et al. believe that NEUROG1 methylation can be used as a sensitive marker for the early screening of colorectal cancer regardless of whether it is CIMP(+) or CIMP(-) colorectal cancer. Therefore, finding markers with higher sensitivity and specificity for CIMP(+) colorectal cancer remains one of the research focuses in the industry. Summary of the Invention

[0005] Based on the above research, the present invention aims to provide new diagnostic markers for CIMP typing of colorectal cancer and also aims to provide new uses of these markers.

[0006] The present invention classifies colorectal cancer into CIMP(+) group and CIMP(-) group according to 16 published CIMP sites, and normal colon tissue is used as a control group. The methylated DNA immunoprecipitation sequencing (MBD-CAP) technique is applied to perform whole-genome methylation sequencing on the three groups of colon tissues. By comparing the differences in promoter CGI methylation values among the three groups, the screening range is narrowed, taking into account both the methylation expression differences and the universality of gene expression. Finally, five genes (LRIF1, FOXL2, KIT, EPB41L4A, B3GALT1) are selected as marker genes for CIMP(+) colorectal cancer for CIMP typing of colorectal cancer. Its advantages are as follows: The CGI methylation levels of these five genes are significantly different, which can be used for CIMP status typing of colorectal cancer, and these five genes take into account functions and are generally expressed in normal colorectal cancer.

[0007] Specifically, the present invention selects five classical CIMP sites such as CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1, and 11 sites confirmed in the literature such as CDKN21, CRABP1, MLH1, CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT21, CDKN2A / ARF, and WRN as standard sites, and uses methylation number / total number ≥ 11 / 16 as the CIMP(+) critical value. The methylated DNA immunoprecipitation sequencing (MBD) protein capture method for methylated DNA fragments is used to perform whole-genome methylation sequencing on normal colorectal tissues, CIMP(-) colorectal cancer, and CIMP(+) colorectal cancer, and compare the promoter CGI methylation differences among them. Five genes (LRIF1, FOXL2, KIT, EPB41L4A, B3GALT1 (new CIMP sites)) are screened from 845 promoter CGI methylation genes unique to CIMP(+) colorectal cancer, and the above genes are verified by pyrosequencing. Compared with classical CIMP sites, these five new sites have higher sensitivity and specificity for CIMP(+) colorectal cancer.

[0008] In the first aspect of the present invention, there is provided the use of a CGI methylation phenotype genome as a marker for CIMP typing of colorectal cancer, wherein the CGI methylation phenotype genome is composed of five genes, namely LRIF1, FOXL2, KIT, EPB41L4A, and B3GALT1.

[0009] In the second aspect of the present invention, there is provided the use of a CGI methylation phenotype genome in the preparation of a reagent or kit for colorectal cancer typing or prognosis diagnosis.

[0010] The diagnostic reagent described above is a reagent for detecting the methylation level of the CGI methylation phenotype genome in a biological sample; the diagnostic kit described above contains a reagent for detecting the methylation level of the CGI methylation phenotype genome in a biological sample.

[0011] The reagent for detecting the methylation level of the CGI methylation phenotype genome in a biological sample is selected from PCR primers with detection specificity for five genes, namely LRIF1, FOXL2, KIT, EPB41L4A, and B3GALT1. The specific sequences are as follows:

[0012]

[0013]

[0014] The biological sample described above is selected from: peripheral blood or tissue samples obtained from a subject.

[0015] In a third aspect of the present invention, there is provided the use of the CGI methylation phenotype genome in the preparation of a therapeutic drug for colorectal cancer. The CGI methylation phenotype genome is a combination of five genes, namely LRIF1, FOXL2, KIT, EPB41L4A, and B3GALT1; the therapeutic drug is a reagent for inhibiting the methylation level of the CGI methylation phenotype genome.

[0016] In a fourth aspect of the present invention, there is provided a colorectal cancer typing or prognosis diagnostic kit, which contains a reagent for detecting the methylation level of the CGI methylation phenotype genome in a biological sample. The CGI methylation phenotype genome is a combination of five genes, namely LRIF1, FOXL2, KIT, EPB41L4A, and B3GALT1.

[0017] Preferably, the diagnostic kit includes a primer system, an amplification system, and a bisulfite modification system. The primer system includes PCR primers as shown in SEQ ID NO.1-10.

[0018] In a fifth aspect of the present invention, there is provided a method for diagnosing colorectal cancer CIMP typing using the above diagnostic kit. The overall detection idea is as follows: first extract DNA from a tissue sample or free DNA (cfDNA) in serum, then perform PCR amplification and pyrosequencing, and subsequently perform data analysis through PyroMarkQ96Advanced software to obtain the methylation beta value of the measured sequence. The primers used for PCR amplification and pyrosequencing are the same, except that the downstream primer is labeled with biotin during pyrosequencing.

[0019] The beneficial guarantees and effects of the present invention are as follows:

[0020] The detection of the CGI methylation phenotype genome is essentially a quantitative PCR detection of blood genome, which has the characteristics of simple operation, high sensitivity, good specificity, high repeatability, etc. Nowadays, it has been more and more widely used in clinical testing technologies and can accurately quantify specific nucleic acid molecules in various samples.

[0021] By detecting the methylation levels of LRIF1, FOXL2, KIT, EPB41L4A, and B3GALT1 in blood or tissue samples, patients with colorectal cancer CIMP(+) and CIMP(-) can be effectively distinguished, and prognosis judgment and treatment can be carried out based on the typing diagnosis results. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the methylation difference of the promoter of the new CIMP locus in three types of tissues: normal colorectal tissue, CIMP(-) colorectal cancer, and CIMP(+) colorectal cancer. DETAILED DESCRIPTION OF THE INVENTION

[0023] Now, in combination with the embodiments and the drawings, the present invention will be described in detail, but the implementation of the present invention is not limited thereto.

[0024] The reagents and raw materials used in the present invention are all commercially available or can be prepared according to the methods in the literature. For the experimental methods without specific conditions noted in the following examples, they are usually carried out under conventional conditions such as those described in "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989) by Sambrook et al., or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by volume.

[0025] I. DNA Extraction

[0026] 1. Tissue DNA Extraction

[0027] For tissue DNA extraction, a genomic DNA purification kit DP329 (Tiangen; Beijing, China) is used, and the steps are as follows:

[0028] (1) Take 50 - 100 mg of tissue in a sample tube, lyse it into a cell suspension, centrifuge at 8960 g for 1 minute, discard the supernatant, and suspend it by shaking with 200 μl of buffer GA (when fresh tumor specimens cannot be detected immediately, they should be stored in a -80 °C refrigerator);

[0029] (2) Add proteinase K and mix well, incubate at 56 °C until the tissue is dissolved, briefly centrifuge to remove the water droplets on the inner wall of the tube cap (note: the lysis time of different tissues is different, usually it can be completed in 1 - 3 hours, and the sample is inverted and mixed 2 - 3 times per hour, or it can also be mixed with a water bath shaker);

[0030] (3) Add 200 μl of buffer GB into the sample tube, invert it thoroughly to mix well, incubate at 70 °C for 10 minutes. After the solution becomes clear, briefly centrifuge to remove the water droplets on the inner wall of the tube cap (Note: White precipitate may form when adding buffer GB, which generally disappears during incubation at 70 °C and will not affect subsequent experiments. If the solution does not become clear, it indicates incomplete cell lysis, which may lead to low DNA extraction yield and impure extracted DNA).

[0031] (4) Add 200 μl of absolute ethanol, shake it vigorously for 15 seconds. Flocculent precipitate may appear at this time. Briefly centrifuge to remove the water droplets on the inner wall of the tube cap.

[0032] (5) Transfer the solution and the flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed in the collection tube), centrifuge at 19158 g for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0033] (6) Add 500 μl of buffer GD into the adsorption column CB3 (please check whether absolute ethanol has been added before use), centrifuge at 19158 g for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0034] (7) Add 700 μl of wash buffer PW into the adsorption column CB3 (please check whether absolute ethanol has been added before use), centrifuge at 19158 g for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0035] (8) Add 500 μl of wash buffer PW into the adsorption column CB3, centrifuge at 19158 g for 30 seconds, and pour out the waste liquid.

[0036] (9) Put the adsorption column CB3 back into the collection tube, centrifuge at 19158 g for 2 minutes, and pour out the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual wash buffer in the adsorption material (Note: The purpose of this step is to remove the residual wash buffer in the adsorption column. The residual ethanol in the wash buffer will affect subsequent enzyme experiments).

[0037] (10) Transfer the adsorption column CB3 into a clean centrifuge tube, suspend and add 50 - 200 μl of elution buffer TE to the middle part of the adsorption membrane, incubate at room temperature for 2 - 5 minutes, centrifuge at 19158 g for 2 minutes, and collect the solution into the centrifuge tube (Note: The volume of the elution buffer should not be less than 50 μl. Too small a volume will affect the recovery efficiency). Measure the concentration of DNA (at 260 nm) with a spectrophotometer. The optical density ratio at 260 / 280 nm should be between 1.8 - 2.0. Otherwise, re - extract (If the extracted DNA product cannot be detected immediately, store it at - 20 °C to prevent degradation).

[0038] 2. Extraction of cell - free DNA (cfDNA) in serum

[0039] Serum cfDNA was extracted using the Free Nucleic Acid Extraction Kit DP339 (Tiangen; Beijing, China), and the steps were as follows:

[0040] (1) Take 300 μl of serum, add 3 μl of 20 mg / ml proteinase K, and mix by gently swirling up and down until the liquid becomes viscous. Incubate in a water bath at 50 °C for 3 h. During the incubation, gently swirl the reaction mixture up and down several times to mix it evenly;

[0041] (2) After the above reaction mixture has cooled to room temperature, add 300 μl of Tris-saturated phenol (pH = 8.0) solution and gently swirl the centrifuge tube up and down for 5 - 10 min until the aqueous and phenolic phases are mixed into an emulsion. Centrifuge at 5000 g for 15 min, carefully aspirate the upper viscous aqueous phase, and transfer it to another centrifuge tube. Repeat this phenol extraction step once;

[0042] (3) Add 300 μl of chloroform - isoamyl alcohol (24:1), mix by swirling up and down, centrifuge at 5000 g for 15 min, and carefully aspirate the upper viscous aqueous phase with a pipette and transfer it to another centrifuge tube. Repeat this step once;

[0043] (4) Add 60 μl of 3 mol / L NaAc and 600 μl of pre - cooled absolute ethanol, gently shake the centrifuge tube at room temperature, and milky white flocculent DNA will appear. Carefully pick up the flocculent DNA with a glass rod and transfer it to another 1.5 ml centrifuge tube; (If no visible precipitate appears, centrifuge at 7500 g for 10 minutes and discard the supernatant)

[0044] (5) Add 100 μl of 70% ethanol, centrifuge at 5000 g for 5 min to wash the DNA, discard the supernatant to remove the residual salt. Repeat once. Volatilize the residual ethanol at room temperature, but do not let the DNA dry completely. Add 30 μl of TE solution to dissolve the DNA and store it at - 20 °C.

[0045] II. Bisulfite modification of genomic DNA

[0046] DNA bisulfite modification was performed using the DNA Methylation Kit (Zymo Research; California, USA.), and the steps were as follows:

[0047] (1) Add 2 μg of DNA to a 1.5 ml EP tube and dilute it to 50 μl with double - distilled water (DDW, autoclaved, the same below);

[0048] (2) Add 5.5 μl of freshly prepared 3 M NaOH and incubate in a water bath at 42 °C for 30 min;

[0049] (3) Add 30 μl of 10 M hydroquinone (the solution turns light yellow) and 520 μl of 3.6 M sodium bisulfite successively;

[0050] (4) Wrap the EP tube with aluminum foil paper, avoid light, and gently invert and mix the solution;

[0051] (5) Add 200 μl of paraffin oil to prevent evaporation and oxidation, and incubate in a water bath at 50 °C in the dark for 16 h.

[0052] III. Purification and recovery of modified DNA

[0053] For the purification and recovery of modified DNA, use the DNA purification and recovery system A7280 (Promega; Beijing, China)

[0054] (1) Insert the pipette tip under the paraffin oil layer, and aspirate the mixture into a clean and sterile 1.5 ml EP tube (sterilized by high-pressure steam, the same below);

[0055] (2) The following uses the DNA purification and recovery system A7280

[0056] 1) Preheat DDW in a water bath at 70 °C, prepare 80% isopropanol, add 1 ml of Promega’s Wizard DNAClean-up resin, gently invert and mix to allow the DNA to fully bind to the resin;

[0057] 2) After tightly connecting the 5 ml syringe barrel to the recovery column provided in the kit, transfer the above mixture to the syringe barrel with a pipette, and use a 2 ml or more EP tube to collect the waste liquid from the column. Add the plunger and gently press to squeeze out the liquid, and a white resin deposit can be seen in the column;

[0058] 3) After separating the syringe from the column, pull out the plunger, then reconnect the syringe barrel to the column, add 2 ml of 80% isopropanol to the syringe barrel, insert the plunger, and gently press to squeeze out the isopropanol;

[0059] 4) Separate the syringe from the column, place the column on a 1.5 ml EP tube, centrifuge at 15364 g for 2 min to dry the resin, remove the column and place it on another 1.5 ml EP tube, add 50 μl of preheated DDW with a pipette, and let it stand at room temperature for 5 min;

[0060] 5) Centrifuge at 15364 g for 20 s to elute the modified DNA solution into a 1.5 ml EP tube, and the final volume is 50 μl;

[0061] (3) Add 5.5 μl of freshly prepared 3 M NaOH and equilibrate at room temperature for 15 min;

[0062] (4) Add 33 μl of 10 M ammonium acetate to neutralize NaOH and adjust the pH of the solution to approximately 7.0;

[0063] (5) Add 4 μl of 10 mg / ml glycogen precipitation indicator to prevent the recovery product from being aspirated when aspirating the residual ethanol;

[0064] (6) Add 270 μl of ice-cold absolute ethanol, place at -20 °C, and precipitate overnight for at least 6 hours;

[0065] (7) Centrifuge at 15364 g for 30 min at 4 °C, discard the supernatant, and collect the precipitate (do not aspirate completely);

[0066] (8) Add 500 μl of 70% ethanol, gently tilt the EP tube, rotate one circle, centrifuge at 15364 g for 5 min at 4 °C, then discard the supernatant, and repeat this step once.

[0067] (9) Centrifuge briefly at room temperature to collect the adhering ethanol at the bottom of the EP tube, carefully aspirate the residual liquid with a pipette, dry at room temperature for 5 min, or when the precipitate changes from opaque to translucent or transparent, add 20 μl of DDW to dissolve the precipitate, and the modified DNA solution is obtained (if the next experiment cannot be carried out immediately, store at -80 °C).

[0068] IV. PCR Amplification

[0069] 1. Instruments and Reagents

[0070] The instrument used for PCR amplification is the real-time quantitative PCR instrument ABI7500 (Applied Biosystems; USA).

[0071] 2. Primers

[0072] The primer sequences are shown in Table 1. The primers were designed according to the primer design principles on the primer design website of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences: https: / / www.urogene.org / index.html.

[0073] Table 1 Primer Sequences for PCR Amplification

[0074]

[0075] 3. PCR Reaction

[0076] Using the bisulfite-treated DNA as a template, perform PCR amplification reaction according to the following reaction system:

[0077] Table 2 PCR Amplification Reaction System

[0078]

[0079] Aliquot the prepared 23 μl mixture into an appropriate number of 0.2 ml (PCR) tubes;

[0080] Add 2 μl of molecular biology grade water to the blank control tube, and add 1 μl of sodium bisulfite-converted DNA to the remaining tubes. Slowly pipette the reaction solution and mix well to evenly distribute the bisulfite-treated DNA into each PCR tube;

[0081] Gently centrifuge the liquid on the wall of the reaction tube and load it into the PCR instrument. Program the PCR instrument according to the steps in Table 3 below:

[0082] Table 3 PCR Amplification Conditions Program

[0083]

[0084] After PCR is completed, store the product at -20 °C for future use.

[0085] V. Pyrosequencing

[0086] The instrument used is the PyroMark Q96 ID pyrosequencer, and the kit used is the PyroMark Gold Q96 Reagents (QIAGEN; Dusseldorf, Germany). The pyrosequencing primer sequences are shown in Table 4.

[0087] Table 4 Pyrosequencing Primer Sequence Table

[0088]

[0089] The specific detection steps are as follows:

[0090] (1) For each reaction, add 40 μl of binding buffer, 29 μl of molecular biology grade water, and 1 μl of magnetic beads to prepare a magnetic bead mixture. Multiply each volume by the number of samples plus 1 to obtain the final volume.

[0091] (2) Add 70 μl of this mixture to 22 wells of a 96-well PCR plate. Leave the last 2 wells empty as negative controls without adding the magnetic bead mixture.

[0092] (3) Add 10 μl of the PCR product obtained in step 1.4.6 to the corresponding wells of the 96-well PCR plate.

[0093] (4) Seal the microwells and then shake at 1400 rpm for at least 15 minutes.

[0094] (5) Add annealing buffer and 20 μl of 1× sequencing primer to 94 wells of a PyroMark Q96 plate.

[0095] (6) Retain the primer control for the last two wells. Add the sequencing primer to one well, and add a mixture of biotinylated primer (2 μl) and sequencing primer (20 μl) to the other well.

[0096] (7) Place the PyroMark Q96 plate on the vacuum preparation workstation and fill all the trays at the workstation with the corresponding solutions.

[0097] (8) Prepare the template on the PyroMark Q96 Advanced software.

[0098] (9) Check the volume of each nucleotide, enzyme mixture, and substrate mixture added to the cartridge. Add them to the cartridge and place it on the PyroMark Q96 Advanced instrument.

[0099] (10) Turn on the vacuum switch in the aspiration probe, place it on the main tray with distilled water, and aspirate approximately 70 ml.

[0100] (11) Take out the 96-well PCR plate, cover it, and place it on the vacuum preparation workstation.

[0101] (12) Place the adsorption probe into the wells of the PCR plate, turn on the vacuum, and carefully capture all the solution from the wells for about 15 s.

[0102] (13) Wash the beads / samples with 70% ethanol for 5 s; continue to add the denaturing solution and rinse for 5 s.

[0103] (14) Aspirate on the rinse buffer tray for 10 s, and then tilt the aspiration probe at an angle of more than 90° for a few seconds.

[0104] (15) Place the vacuum aspiration probe on top of the PyroMark Q96 plate without touching the liquid.

[0105] (16) Turn off the vacuum and lower the handle on the PyroMark Q96 well plate.

[0106] (17) Gently shake the aspirated probe from the side for 30 - 60 s to release the magnetic beads on the PyroMark Q96 plate containing the sequencing primer.

[0107] (18) Rinse the vacuum adsorption probe on the rinse tray containing distilled water and stir for 10 s.

[0108] (19) Aspirate 70 ml of distilled water on the main tray.

[0109] (20) Vertically move the aspiration probe at an angle of more than 90° for a few seconds, and then disconnect the vacuum.

[0110] (21) Place the PCR plate on the metal tray and heat it at 80 °C for 5 min.

[0111] (22) After an interval of 30 s, place the sequencing plate on the PyroMark Q96 Advanced instrument, cover it, and start running.

[0112] (23) After the run is completed, invert the PyroMark Q96 well plate and rinse the drug column with deionized water.

[0113] (24) Fill each well of the drug column with water and force the water through the small pinhole at the bottom by applying pressure with a finger to the top.

[0114] (25) Repeat step (24) for each well at least 3 times.

[0115] (26) Let it dry at room temperature.

[0116] (27) Take out all the trays from the vacuum preparation workstation, rinse them with deionized water, and dry them at room temperature; perform data analysis through the PyroMark Q96 Advanced software to obtain the methylation β value of the measured sequence. The results are shown in Figure 1 : The new CIMP sites LRIF1, FOXL2, KIT, EPB41L4A, and B3GALT1 are generally expressed in normal colorectal cancer. The methylation differences of the promoters in the three types of tissues, namely normal colorectal tissue, CIMP(-) colorectal cancer, and CIMP(+) colorectal cancer, are significant, and the CIMP status of colorectal cancer can be typed.

[0117] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can also make various equivalent variations or substitutions without departing from the spirit of the present invention. These equivalent variations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Use of a reagent for detecting a CGI methylation phenotype gene combination in a biological sample in the preparation of a colorectal cancer typing diagnostic kit, characterized in that, The colorectal cancer is classified into CIMP(-) and CIMP(+) types. The CGI methylation phenotype gene combination is a combination of five genes: LRIF1, FOXL2, KIT, EPB41L4A, and B3GALT1. The reagent is selected from PCR primer pairs that are specific for detecting the above genes. The primer pairs for the LRIF1 gene are shown as SEQ ID NO.1 and SEQ ID NO.2; the primer pairs for the FOXL2 gene are shown as SEQ ID NO.3 and SEQ ID NO.4; the primer pairs for the KIT gene are shown as SEQ ID NO.5 and SEQ ID NO.6; the primer pairs for the EPB41L4A gene are shown as SEQ ID NO.7 and SEQ ID NO.8; the primer pairs for the B3GALT1 gene are shown as SEQ ID NO.9 and SEQ ID NO.

10.

2. The application according to claim 1, characterized in that, The downstream primer in the primer pair is labeled with a biofluorescent molecule.

3. The application according to claim 1, characterized in that The biological sample is obtained from the peripheral blood or tissue specimen of the subject.

4. A colorectal cancer typing diagnosis kit, characterized in that, The kit contains reagents for detecting the methylation degree of the CGI methylation phenotype gene combination in the biological sample. The CGI methylation phenotype gene combination is a combination of five genes: LRIF1, FOXL2, KIT, EPB41L4A, and B3GALT1. The diagnostic kit includes a primer system, an amplification system, and a bisulfite modification system. The primer system is composed of PCR primers shown as SEQ ID NO.1 to 10. The colorectal cancer is classified into CIMP(-) and CIMP(+) types.

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