Gene marker combination for diagnosing or predicting colorectal cancer and application thereof

Abstract

The invention discloses a gene marker combination for diagnosing and predicting colorectal cancer and application of the gene marker combination, and belongs to the technical field of cancer molecular diagnosis. The CASZ1, C1QL3 and PRRX1 genes are used as a methylation gene marker combination for early diagnosis or prediction of the colorectal cancer, and choices of technicians in the field are enriched. The methylation levels of CASZ1, C1QL3 and PRRX1 are detected by adopting a methylated DNA co-immunoprecipitation technology, early diagnosis or prediction of colorectal cancer can be realized, the sensitivity is high, the specificity is strong, and the kit has very important clinical application value.

Description

Technical Field

[0001] The present application relates to the technical field of cancer molecular diagnosis, and in particular to a combination of gene markers for diagnosing and predicting colorectal cancer and its application. Background Art

[0002] CRC (Colorectal cancer, CRC) is a common malignant tumor, which refers to a malignant tumor that occurs in the colon or rectum of the lower digestive tract of the human body. It is one of the major cancers that threaten human health. In recent years, the incidence and mortality of CRC in my country have been on the rise. It can be seen that the current work of CRC prevention, diagnosis and treatment is arduous and long-term, and the early diagnosis, treatment and screening of CRC are of great significance.

[0003] According to the multi-stage theory of carcinogenesis, the occurrence of CRC is morphologically manifested as a staged evolution from normal mucosal hyperplasia, polyp adenoma formation, adenoma carcinoma to invasive metastasis. It takes 10-15 years for polyp adenoma to evolve into CRC. The cure rate of CRC detected in the early stage can reach more than 90%, while the cure rate in the late stage is less than 10%. Therefore, risk identification, screening and early diagnosis of CRC precancerous lesions are very necessary to reduce the mortality rate of CRC.

[0004] The "CRC and Early Diagnosis and Treatment Program" published in 2024 mentioned that CRC precancerous lesions include adenomas with a diameter ≥10mm, adenomas with a villous structure ≥25% (i.e., villous adenomas or mixed adenomas), and other lesions with high-grade intraepithelial neoplasia. It is recommended that all adenomas, polyps, especially CRC precancerous lesions and CRC patients receive standardized treatment as soon as possible.

[0005] Colonoscopy is the gold standard for colorectal cancer screening, but due to its high invasiveness and cumbersome intestinal preparation, compliance with colonoscopy screening is low. In addition, there is a large demand for colonoscopy. With the aging of the population, the number of elderly people and people over 40 years old is increasing, and the number of people undergoing colonoscopy has shown a blowout growth. The large-scale use of colonoscopy for screening will also cause a huge waste of resources. The traditional screening program adopts a two-step screening model combining a questionnaire survey with two fecal occult blood tests (FIT). Anyone who is positive in any one of the items is judged as a positive in the initial screening, indicating that they are at high risk and need to undergo a colonoscopy. This screening has problems such as too high false positives and low colorectal cancer detection rate, and this screening model has resulted in insufficient manpower investment in the hospital, so the project is progressing slowly.

[0006] The current marketed products of colorectal cancer auxiliary diagnosis technology based on blood DNA detection generally have low sensitivity, less than 85%, which cannot meet clinical needs. Clinical practice shows that the main limitation of blood Septin9 methylation detection is that its sensitivity for identifying colorectal cancer and precancerous lesions (adenomas) is relatively low, and the sensitivity for advanced adenomas is only 7.9% to 38.7%.

[0007] The colorectal cancer screening technology based on fecal DNA detection mainly targets the characteristics of gene mutations and / or methylation of colorectal exfoliated cells, overcoming the main defects of detecting microbleeding. There are single-target and multi-target schemes, and it can also be combined with FIT for detection. It has the advantages of no need for special equipment, no dietary restrictions, and non-invasiveness. Although there have been significant improvements in the sensitivity and specificity of colorectal cancer, the detection rate for 0-II early cancers is still below 90%, and the detection rate of advanced adenomas such as precancerous lesions and high-grade intraepithelial neoplasia is even lower, all less than 65% or even lower. Although fecal DNA testing can be achieved by sampling at home, fecal sampling is relatively private and inconvenient, which is different from the public's medical habits. It will take a very long time for the public to accept fecal DNA testing for health education, so there are still certain problems with testing compliance in the screening process.

[0008] Therefore, developing new colorectal cancer markers with higher sensitivity based on blood samples with the highest clinical user compliance can become an urgent problem to be solved. Summary of the invention

[0009] In order to solve at least one of the above technical problems, the technical solution adopted in this application is as follows.

[0010] In a first aspect, the present application provides the use of a detection reagent for the methylation level of a gene marker combination in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer, wherein the gene marker combination includes CASZ1, C1QL3 and PRRX1.

[0011] Almost all tumors are caused and promoted by genetic changes and epigenetic variations. By comparing tumor cells and normal cells, a large number of epigenetic abnormalities have been discovered and reported, among which DNA methylation is the most common epigenetic effect.

[0012] Methylation of cytosine on DNA is a covalent "acquired" modification of DNA. DNA methylation is carried out by DNA cytosine methyltransferases (DNMTs). DNMTs can transfer a methyl group from an S-adenosylmethionine to the C-5 position of cytosine. DNA methylation occurs almost specifically at the CpG doublet position. CpG doublets are unevenly distributed in the human genome, and the concentrated and enriched areas are generally called CpG islands. Such CpGs exist in the repetitive sequences of the human genome and in the regulatory regions at the 5' end of many genes. DNA methylation abnormalities that occur in tumors include hypomethylation (or demethylation) and hypermethylation. The occurrence of hypermethylation includes but is not limited to tumor suppressor genes, and the occurrence of hypomethylation includes but is not limited to proto-oncogenes.

[0013] All aspects of tumor development and progression may be related to DNA methylation changes, involving cell cycle regulation, DNA damage repair, biochemical metabolism of carcinogenic compounds, apoptosis and angiogenesis. Different types of tumors may have a specific group of tumor suppressor genes that are highly methylated and proto-oncogenes that are hypomethylated, that is, there is a specific cancer methylation map, and the type of cancer may be confirmed based on the methylation map.

[0014] In the present application, the inventors unexpectedly discovered, after long-term exploration and verification of a large number of clinical samples, that the methylation levels of CASZ1, C1QL3 and PRRX1 genes differ significantly between colorectal cancer and non-colorectal cancer.

[0015] In some embodiments of the present application, the detection reagent includes a methylated region enrichment or methylation treatment reagent and also includes a qPCR detection reagent.

[0016] In some embodiments of the present application, the methylation regions of the CASZ1, C1QL3 and PRRX1 genes are obtained in colorectal cancer population samples and normal samples using a probe combination, and the coverage information of each probe is as follows:

[0017] The methylated region of the CASZ1 gene is chr1:10647750-10648000, the primer pair is shown in SEQ ID No. 1 and SEQ ID No. 2, and the probe is shown in SEQ ID No. 3; the methylated region of the C1QL3 gene is chr10:16520568-16520767, the primer pair is shown in SEQ ID No. 4 and SEQ ID No. 5, and the probe is shown in SEQ ID No. 6; the methylated region of the PRRX1 gene is chr1:170661257-170661457, the primer pair is shown in SEQ ID No. 7 and SEQ ID No. 8, and the probe is shown in SEQ ID No. 9.

[0018] Methylation enrichment technology is an analytical method used to study methylation modifications on DNA. DNA methylation is an important epigenetic modification that involves the addition of methyl groups to the cytosine ring in DNA molecules. This modification plays a key role in regulating biological processes such as gene expression, cell differentiation, and genome stability. Therefore, understanding the state of DNA methylation is very important for understanding biological processes and the occurrence and development of diseases.

[0019] Common methylation enrichment techniques include methylation-specific PCR (MSP), methylation-sensitive restriction enzyme digestion, MeDIP-Seq (Methylated DNA Immunoprecipitation sequencing) and MBD-Seq (Methyl-CpG Binding Domain sequencing), among which: MSP uses methylation-specific primers to selectively amplify methylated DNA fragments through PCR. It is simple and rapid, suitable for the analysis of specific CpG sites, but cannot provide methylation information for the entire genome and is only applicable to pre-determined target regions.

[0020] Methylation-sensitive restriction enzyme cutting uses the difference in the sensitivity of restriction enzymes to DNA sequences to distinguish between methylated and unmethylated DNA regions. This method is based on the principle that DNA methylation affects the sensitivity of bases on the cytosine ring to restriction enzymes. This technology does not require the use of expensive sequencing technology and can be analyzed by methods such as gel electrophoresis. However, it cannot provide high-resolution information on a single CpG site, and usually provides the methylation status of the entire region. At the same time, due to the specificity of the selected restriction enzyme, some methylation sites may be missed or over-detected. In addition, it is not possible to directly distinguish between 5-methylcytosine and other forms of DNA modification.

[0021] MeDIP-Seq uses methylated DNA antibodies to selectively enrich methylated DNA fragments, and then analyzes the enriched products through high-throughput sequencing technology. It can enrich the entire methylated genomic region and is suitable for whole-genome methylation analysis. However, it cannot provide high-resolution information on a single CpG site.

[0022] MBD-Seq uses methylated DNA binding proteins (such as MBD2 or MBD3) to enrich methylated DNA fragments, which are then analyzed by sequencing. It can provide high enrichment efficiency and is suitable for genome-wide methylation analysis. However, similar to MeDIP-Seq, it cannot provide high-resolution information on a single CpG site.

[0023] In this application, the methylation treatment is also referred to as methylation conversion. Common methylation treatment sequencing technologies include Bissulfite sequencing (BS-seq). BS-seq uses sulfite to treat DNA, converting non-methylated cytosine to uracil, while methylated cytosine is not affected, and then analyzing by sequencing. It can provide high-resolution information of a single CpG site and can perform methylation analysis on the whole genome, but the experimental steps are cumbersome.

[0024] In some embodiments of the present application, methylated DNA immunoprecipitation (MeDIP) technology is used to enrich methylated fragments. The methylated DNA antibody is selected from one of 5-methylcytidine antibody, 5-methylcytosine (5-mC) antibody, 5-hydroxymethylcytosine (5-hmC) antibody, 5-formylcytosine (5-fC) antibody, and 5-carboxycytosine (5-caC) antibody.

[0025] In some preferred embodiments of the present application, the methylated region enrichment reagent includes a 5-methylcytosine antibody.

[0026] In other embodiments of the present application, the methylation treatment agent includes bisulfite.

[0027] Furthermore, the qPCR detection reagent includes a primer pair and a probe targeting the methylated sequence.

[0028] Furthermore, the methylated region of the CASZ1 gene is chr1:10647750-10648000, the primer pairs targeting the sequence after methylation treatment are shown in SEQ ID No. 10 and SEQ ID No. 11, and the probe is shown in SEQ ID No. 12; the methylated region of the C1QL3 gene is chr10:16520568-16520767, the primer pairs targeting the sequence after methylation treatment are shown in SEQ ID No. 13 and SEQ ID No. 14, and the probe is shown in SEQ ID No. 15; the methylated region of the PRRX1 gene is chr1:170661257-170661457, the primer pairs targeting the sequence after methylation treatment are shown in SEQ ID No. 16 and SEQ ID No. 17, and the probe is shown in SEQ ID No. 18.

[0029] It is possible to achieve early detection of cancer by performing relevant methylated DNA analysis on a certain type of cancer. The current mainstream methylation analysis method is bisulfite treatment, which includes denaturation, deamination and desulfonation. The DNA is first denatured into a single strand, and then subjected to high temperature, high salt, acidic and alkaline environments, encountering two extremes of ice and fire. The morphology of the converted DNA obtained is: mainly single strands, double strands mixed, fragment nicks, gap damage, and uracil state nucleotides. This process generally causes 90% of the DNA template to be lost, and a large amount of methylation information cannot be detected by subsequent processes. At the same time, during the base conversion treatment, there are incomplete or excessive sequence conversion, resulting in artificial bias, which will be further amplified by subsequent PCR amplification, resulting in inaccurate signals. Therefore, the methylation markers currently obtained based on bisulfite treatment generally have the problem of low sensitivity, especially in blood samples. After the limited number of free DNA fragments are treated with bisulfite, the difficulty of detecting methylation levels is greatly increased.

[0030] Although the sensitivity for colorectal cancer can be improved by increasing the number of methylated genes based on bisulfite treatment, for example, the combined detection of 3 colorectal cancer gene methylation by Beijing Aikelun Medical Technology Co., Ltd. was approved by the National Medical Products Administration NMPA in 2022. The sensitivity of clinical trials using blood sample cfDNA treated with bisulfite was 84.75% (328 / 387), but the sensitivity still cannot meet the actual clinical needs. Therefore, trying to discover colorectal cancer-related gene methylation markers in human blood samples based on non-bisulfite treatment methods and effectively detect changes in their methylation levels has also become the most urgent need for early cancer screening.

[0031] The second aspect of the present application provides the use of a detection reagent for the methylation level of a combination of gene markers in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on the following method: S1, obtain cfDNA samples from biological samples of subjects; S2, enriching methylated regions of cfDNA samples or performing methylation treatment; S3, using the product enriched or treated in step S2 as a template, and using primer pairs and probes targeting the methylated regions of the untreated or treated gene marker combination to perform qPCR amplification; If there is a typical amplification curve and the Ct value is not greater than the preset threshold, the test subjects are diagnosed with colorectal cancer or have a risk of colorectal cancer. Among them, the gene marker combination includes CASZ1, C1QL3 and PRRX1, the methylation region of the CASZ1 gene is chr1:10647750-10648000; the methylation region of the C1QL3 gene is chr10:16520568-16520767; and the methylation region of the PRRX1 gene is chr1:170661257-170661457.

[0032] An amplification curve is a graph that detects product accumulation during the polymerase chain reaction (PCR). It is generated by monitoring the increase in fluorescent signal in the reaction solution. The following are the characteristics of a typical PCR amplification curve: Initial stage: Threshold Cycle (Ct) value: In the early stages of a PCR reaction, the fluorescence signal may be low, but as the PCR product accumulates, the fluorescence signal gradually increases. The Ct value refers to the number of cycles required in a PCR reaction for the fluorescence signal to rise above a pre-set threshold. The lower the Ct value, the higher the starting amount of target DNA in the sample.

[0033] Exponential growth phase: Exponential phase: In the middle of the PCR reaction, the PCR product grows exponentially. At this time, the Ct value increases faster, reflecting the exponential growth of the target DNA in the PCR reaction.

[0034] Platform stage: Platform stage: In the later stage of PCR reaction, the accumulation of PCR products reaches saturation and no longer grows exponentially. The PCR amplification curve at this stage forms a platform, and the increase of Ct value becomes slow.

[0035] In some embodiments of the present application, the preset threshold is determined based on a representative value of the Ct value obtained by the same method for a group of non-colorectal cancer samples and / or a group of colorectal cancer samples. The representative value is selected from the mean, mode, median, one-quarter quantile or one-third quantile. In a third aspect, the present application provides a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on DNA immunoprecipitation detection of methylation levels of a gene marker combination, wherein the gene marker combination includes CASZ1, C1QL3 and PRRX1, the methylation region of the CASZ1 gene is chr1:10647750-10648000, the primer pair is as shown in SEQ ID No. 10 and SEQ ID No. 11, and the probe is as shown in SEQ ID No. 12; the methylation region of the C1QL3 gene is chr10:16520568-16520767, the primer pair is as shown in SEQ ID No. 13 and SEQ ID No. 14, and the probe is as shown in SEQ ID No. 15; the methylation region of the PRRX1 gene is chr1:170661257-170661457, the primer pair is as shown in SEQ ID No. 16 and SEQ ID No. 17, and the probe is as shown in SEQ ID Shown in No. 18.

[0036] In some embodiments of the present application, the diagnosis is an early diagnosis, specifically, the early stage is CRC stage 0~I or stage II.

[0037] Compared with the prior art, this application has the following beneficial effects: The present application provides methylation levels of methylation regions of CASZ1, C1QL3 and PRRX1 genes as markers for early diagnosis or prediction of colorectal cancer, enriching the options for those skilled in the art.

[0038] This application uses methylated DNA immunoprecipitation technology to detect the methylation levels of methylated regions of CASZ1, C1QL3 and PRRX1 genes, which has high sensitivity and strong specificity and has very important clinical application value.

[0039] This application is based on the methylation levels of CASZ1, C1QL3 and PRRX1 genes, and can experiment with early detection of colorectal cancer, providing more marker options for early colorectal screening.

[0040] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] By reading the detailed description below with reference to the accompanying drawings, the above and other purposes, features and advantages of the exemplary embodiments of the present application will become readily understood. In the accompanying drawings, several embodiments of the present application are shown in an exemplary and non-limiting manner, wherein: Figure 1 The flowchart of the gene marker combination methylation gene detection in Example 2 of the present application is shown.

[0042] Figure 2 Amplification curves of qPCR detection based on methylated DNA immunoprecipitation enrichment method and qPCR detection based on bisulfite conversion treatment of three genes in Example 2 of the present application are shown, A: qPCR detection of CASZ1 gene based on methylated DNA immunoprecipitation enrichment method; B: qPCR detection of C1QL3 gene based on methylated DNA immunoprecipitation enrichment method; C: qPCR detection of PRRX1 gene based on methylated DNA immunoprecipitation enrichment method; D: qPCR detection of CASZ1 gene based on bisulfite conversion treatment; E: qPCR detection of C1QL3 gene based on bisulfite conversion treatment; F: qPCR detection of PRRX1 gene based on bisulfite conversion treatment.

[0043] Figure 3 The ROC curve (A) of qPCR detection based on the methylated DNA immunoprecipitation enrichment method and the ROC curve (B) of qPCR detection based on bisulfite conversion treatment for 119 samples in Example 2 of the present application are shown. DETAILED DESCRIPTION

[0044] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or disclosure involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0045] In order to make the technical problems, technical solutions and beneficial effects solved by the present application more clearly understood, the present application is further described in detail below in conjunction with embodiments.

[0046] The following examples are used to demonstrate the preferred embodiments of the present application. It will be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present application, and therefore can be considered as preferred embodiments of the present application. However, it should be understood by those skilled in the art based on this specification that many modifications can be made to the specific embodiments disclosed herein, and still the same or similar results can be obtained without departing from the spirit or scope of the present application.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0048] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0049] The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0050] Example 1 Discovery of Colorectal Cancer-Specific Methylated Genes In order to screen for biomarkers that are specifically methylated in colorectal cancer, the inventors collected blood samples and corresponding homologous cancer tissues and adjacent adjacent cancer samples from 548 clinical colorectal cancer patients from the First Affiliated Hospital of Zhejiang University School of Medicine and other central hospitals, as well as blood samples and corresponding tissue samples from 389 clinical colorectal cancer-negative patients. The differentially methylated genes or regions screened in the blood samples must also be verified in the tissue samples to be more reliable clinically significant methylation markers.

[0051] 1. Prepare methylated DNA sample library (1) DNA extraction cfDNA was extracted using a commercial extraction kit according to the instructions.

[0052] Qubit4.0 and Qsep100 were used to control the concentration and fragment distribution of nucleic acids, respectively. The yield of cfDNA extracted from 2 mL of human plasma should be greater than 5 ng, and cfDNA has an enrichment peak at or near 167 bp. When the yield is greater than 50 ng, Qsep100 capillary electrophoresis is used for fragmentation quality control. When there is large fragment contamination, magnetic beads are used for fragment screening to remove large fragments.

[0053] The extraction of genomic DNA from samples of cancer tissue, paracancerous tissue, normal tissue, etc. is performed using conventional commercial kits or self-prepared reagents according to the instructions. Genomic DNA can be fragmented by ultrasound or enzyme digestion to obtain DNA with a length of about 200 bp for convenient library construction.

[0054] (2) Library construction The library construction kits of commercial companies can be used according to the instructions. For example, Rapid Plus DNA LibPrep Kit for illumina (Cat. No. RK20208, ABclonal) or VAHTS UniversalPro DNA Library Prep Kit for illuminaVazyme (Cat. No. ND608-02, Novagene) and other similar kits can be used for end repair, adding "A" tails, and connecting to Adapters. In this example, VAHTS Universal ProDNA Library Prep Kit for illuminaVazyme (Cat. No. ND608-02, Novagene) was used for library construction.

[0055] (3) Methylated DNA co-immunoprecipitation The methylation enrichment of cfDNA and tissue genomic DNA is carried out in different reactions. Each methylation enrichment reaction can complete the mixing of 12 to 100 cfDNA libraries at the same time (the input amount of each cfDNA sample library is about 10ng), and can complete the mixing of 10 to 24 genomic DNA libraries at the same time (the input amount of each genomic DNA library is about 100ng). The methylation enrichment based on the 5mC antibody principle is operated according to the instructions of the methylation enrichment kit of a commercial company or the instructions of the self-prepared reagent. This example uses the zymoMeDIP kit (Cat. No. D5101-A). Then, according to the instructions, the methylation enrichment reaction is purified and 10 to 12 rounds of conventional PCR amplification are performed with universal sequencing primer pairs to obtain a methylated DNA fragment library with a yield of more than 500ng per reaction.

[0056] 2. Prepare DNA probe library (1) Probe design The inventors selected the longest CASZ1, C1QL3, and PRRX1 gene transcripts from the database to confirm the gene locations, and screened and obtained sequence information of the promoter region, 5'UTR, first exon (exon1), and 1kb upstream of the start codon (a total of 9.6Kb).

[0057] Probes were designed for the target methylation regions of the CASZ1, C1QL3, and PRRX1 genes.

[0058] Probe design principles: (1) Full coverage of the target area without gaps; (2) No overlap; (3) Each probe is 120 nt in length.

[0059] A list of specific genomic corresponding positions of the targeted region and a detailed list of specific genomic corresponding positions of the region covered by the probes designed thereby can be obtained by those skilled in the art through the description of this patent specification.

[0060] (2) Probe synthesis Using the above probe design principles and removal criteria, a total of 120 probes were designed for the methylation regions of CASZ1, C1QL3, and PRRX1 genes, covering all possible CpG sites. The coverage region annotations include coding genes and microRNA genes, and the probe coverage information is shown in Table 1.

[0061] Table 1 Probe coverage information

[0062] Customize probe panels at Nanoda (Nanjing) Biotechnology Co., Ltd.

[0063] 3. DNA capture probe hybridization NadPrep hybrid capture reagent (Cat. No.REF1005101, Naonda) was used for liquid phase hybrid capture. The hybrid capture reaction can be single hybrid or multi-hybrid. The total amount of MeDIP amplified library input for each hybrid capture reaction should be between 300ng and 8μg. 500ng of the purified library (if less than 500ng, all of it should be input) was added to Human Cot DNA and NadPrep ® Nano Blockers were placed in a vacuum concentrator preheated to 42°C and dried at 1000 rpm. After drying, the prepared hybridization reaction solution (including the above-mentioned probe panel) was added, and the mixture was centrifuged with shaking for an instant, and hybridization capture was performed for 4-16 hours under the hybridization program: 95°C / 30sec; 65°C / Hold (100°C hot cover). The washed streptavidin magnetic beads were then added to the hybridization system and incubated for 40 minutes, during which time they were vortexed every 10 minutes to ensure that the magnetic beads were completely resuspended. It is worth noting that the reaction temperature for hybridization capture is the conventional 65°C, rather than the 63°C for methylation probes designed based on bisulfite conversion.

[0064] After the hybridization capture reaction is completed, the bound magnetic beads are washed with the four washing solutions provided by the kit. The residual liquid needs to be discarded in each step; finally, 20 μL of nuclease-free water is added and gently vortexed to mix.

[0065] 4. PCR amplification and purification after hybridization capture The products after hybridization capture were amplified by PCR, and the reagents used were the amplification reagents in the VAHTS Universal Pro DNA LibraryPrep Kit for illumina (Cat. No. ND608-02, Vazyme) for the experiment, with 12-13 cycles. After the amplification, the products were purified using an equal volume of VAHTS DNA Clean Beads (Cat. No. N411-03, Vazyme) to obtain a relatively pure hybridization capture library; the library concentration was quantitatively determined using Qubit4.0, and the fragment size of the library was detected using the Qsep100 fully automatic nucleic acid protein analyzer.

[0066] 5. Library sequencing and bioinformatics analysis Dilute the library concentration to be loaded onto the machine to 4nM, mix according to the ratio of the required data volume, and the total data volume cannot exceed 120G. After mixing, take out 5μL of the library, add 5μL of 0.2N NaOH, mix by pipetting, and denature for 5 minutes. Immediately after the end, add 990μL of HT1 Buffer (REF: 15058251, illumina), vortex to mix, take out 105μL and add 1295μL of HT1 Buffer, vortex to mix, and this is the library to be loaded onto the machine, with a concentration of 1.5pM.

[0067] The sequencer is NextSeq 550Dx produced by illumina, and the reagents used are High Output Reagent Cartridge v2 (REF: 15057929, illumina) (300 cycles), High Output Flow Cell Cartridge v2.5 (REF: 20022408, illumina) and Buffer Cartridge v2 (REF: 15057941, illumina). 1300 μL of the library on the machine is added to the sample position of the High Output Reagent Cartridge v2, and each reagent is placed in turn to start sequencing; this example uses double-end sequencing, and the total duration is about 30 hours.

[0068] 6. Quality Control of Sequencing Data Fastp (version 0.22.0) was used to perform quality control on the offline data to remove low-quality bases. The overall Q20 of cleandata was above 90%, and the Q30 was above 85%. The average sequencing depth was about 300×. The average on-target rate of the probes in the above probe combination was above 80%, which shows that the detection of cancer-related methylation regions based on the combination of methylation immunoprecipitation and liquid phase hybridization capture probes in this embodiment is feasible and effective.

[0069] 7. Analysis of differentially methylated regions of CASZ1, C1QL3 and PRRX1 genes related to colorectal cancer The DiffBind tool (version 3.8.4) was used to screen differential peaks between tumors and non-tumors. The DESeq and EdgeR algorithms were used to select the intersection and prioritize the areas within the panel.

[0070] In this example, the characteristic methylation regions with the most significant differences between the colorectal cancer group and the non-colorectal cancer group were screened out from the CASZ1, C1QL3, and PRRX1 gene regions covered by 120 probes.

[0071] For the CASZ1 gene, the methylation region with the most significant difference in methylation levels was chr1:10647750-10648000.

[0072] For the C1QL3 gene, the methylation region with the most significant difference in methylation levels was chr10:16520568-16520767.

[0073] For the PRRX1 gene, the methylation region with the most significant difference in methylation levels was chr1:170661257-170661457.

[0074] The RPM index (Reads per million mapped reads) of this characteristic methylation difference region was analyzed in 548 colorectal cancer positive samples and 389 normal healthy population samples. The P value was 0.005, indicating a significant difference.

[0075] Example 2 Detection of CASZ1, C1QL3 and PRRX1 methylation genes in clinical samples and comparison of different treatment schemes In order to further verify the clinical performance of the differentially methylated regions of the colorectal cancer-related CASZ1, C1QL3 and PRRX1 genes on CRC plasma samples, the inventors used the qPCR method to detect 61 plasma samples clinically diagnosed with CRC and 58 plasma control samples that were negative for colonoscopy. Among the above 61 plasma samples clinically diagnosed with CRC, there were 11 samples of CRC stage 0~I, 12 samples of CRC stage II, 18 samples of CRC stage III, and 20 samples of CRC stage IV.

[0076] The flow chart of methylation level detection of CASZ1, C1QL3 and PRRX1 genes is as follows Figure 1 As shown, specifically: (1) DNA extraction cfDNA was extracted using a commercial extraction kit according to the instructions.

[0077] Qubit4.0 and Qsep100 were used to control the concentration and fragment distribution of nucleic acids, respectively. The yield of cfDNA extracted from 4 mL of human plasma should be greater than 10 ng, and cfDNA has an enrichment peak at or near 167 bp. When the yield is greater than 50 ng, Qsep100 capillary electrophoresis is required for fragmentation quality control. When there is large fragment contamination, magnetic beads are used for fragment screening to remove large fragments.

[0078] (2) Methylated DNA treatment ① Methylated DNA co-immunoprecipitation Take 1 / 2 of the total amount of nucleic acid extracted above for cfDNA methylation enrichment, which is carried out in different reactions. The methylation enrichment based on the 5mC antibody principle uses the zymoMeDIP kit (Cat. No. D5101-A). The methylation enrichment reaction is purified according to the instructions, and the elution volume is 50 μL.

[0079] ② Bisulfite conversion of methylated DNA Take 1 / 2 of the total amount of nucleic acid extracted above and perform bisulfite treatment for cfDNA methylation, which is divided into different reactions. The methylated DNA treatment based on the bisulfite conversion principle uses the DNA conversion kit (EZ DNA Methylation Kit, D5002) of ZYMO RESEARCH Biological Company to perform bisulfite treatment of DNA. The elution volume is 50 μL.

[0080] (3) qPCR detection The primers and probes were synthesized at Shanghai Bio-Tech Co., Ltd. The specific sequence information is as follows: The sequences of the primer pairs for Taqman MGB probes for methylated DNA immunoprecipitation enrichment are shown in Table 2.

[0081] Table 2 Primer pairs for Taqman MGB probe enrichment of methylated DNA immunoprecipitation

[0082] The 3' end of the probe was labeled with MGB.

[0083] PCR amplification was performed using methylated DNA enriched by immunoprecipitation as a template, and the final concentration of each primer was 10 μM. The PCR reaction system was 5 μL of enriched template DNA, including 2.5 μL of the premix of the above primers; 17.5 μL of PCR reaction solution reagent (2×Rapid Taq Master Mix), and the total volume was supplemented with water to 35 μL. The PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 15 seconds, 60°C for 40 seconds, and 48 cycles of amplification.

[0084] The sequences of the Taqman MGB probe primer pairs after bisulfite conversion are shown in Table 3.

[0085] Table 3 Taqman MGB probe primer pairs after bisulfite conversion

[0086] The 3' end of the probe was labeled with MGB.

[0087] PCR amplification was performed using bisulfite-converted DNA as a template, with a final concentration of 10 μM for each primer. The PCR reaction system consisted of 5 μL of enriched template DNA, 2.5 μL of the premix of the above primers, 17.5 μL of PCR reaction solution reagent (2×RapidTaq Master Mix), and the total volume was filled with water to 35 μL. The PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 15 seconds, 60°C for 40 seconds, and 48 cycles of amplification.

[0088] (4) Analysis of clinical sample test results The offline data were analyzed, and 119 samples were enriched by methylated DNA immunoprecipitation method and bisulfite treatment DNA method and then tested by qPCR.

[0089] Figure 2Examples of qPCR amplification curves of samples from the same CRC patient (sample number: 103492) using the methylated DNA immunoprecipitation enrichment method and the bisulfite treatment DNA method are shown. Figures AC represent the qPCR amplification curves of the three genes CASZ1, C1QL3, and PRRX1 after methylated DNA immunoprecipitation enrichment, with Ct values ​​of 35.78, 35.93, and 34.88, respectively. Figures DE represent the qPCR amplification curves of bisulfite treatment DNA, with Ct values ​​of 38.50, 38.86, and 36.14, respectively. The use of methylated DNA immunoprecipitation enrichment followed by qPCR detection has obvious advantages.

[0090] The Ct value of the sample with the detection result of Undetermined is set to 45, and the logistic regression formula is used for calculation. The ROC curve is drawn according to the calculation results, as shown in the figure below: Figure 3 As shown in the figure, the area under the curve (AUC) of the ROC curves obtained based on the two different methods are 0.987 and 0.942, respectively. According to the ROC curve, the cut-off values ​​are set for different methods: based on the methylated DNA immunoprecipitation enrichment method qPCR detection, the cut-off value is set to logistic scores = 800; based on the bisulfite conversion treatment qPCR detection, the cut-off value is set to logistic scores = 700. If the three gene amplification logistic scores of the sample being tested are equal to or lower than the set cut-off value, the sample is judged as a negative sample, otherwise it is judged as positive. Thus, the test results of 119 samples were statistically analyzed. The formula is as follows: logistic scores = e k / (1+e k ) Methylated DNA immunoprecipitation: k=-1.418*CtCASZ1+-0.638*CtC1QL3+-1.255*CtPRRX1+123.867 Bisulfite conversion: k=-0.686*CtCASZ1+-0.317*CtC1QL3+-0.152*CtPRRX1+43.411 Table 4 shows the comparison between qPCR detection based on methylated DNA immunoprecipitation enrichment method and colonoscopy results (gold standard), Table 5 shows the comparison between qPCR detection results based on bisulfite conversion treatment and colonoscopy results, and Table 6 shows the comparison between qPCR detection based on methylated DNA immunoprecipitation enrichment method and qPCR detection results based on bisulfite conversion treatment.

[0091] Table 4 Comparison of qPCR test results based on methylated DNA immunoprecipitation enrichment method with colonoscopy results

[0092] Table 5 qPCR test results based on bisulfite conversion treatment compared with colonoscopy results

[0093] Table 6 Comparison of enrichment methods based on methylated DNA immunoprecipitation and bisulfite conversion

[0094] As shown in Tables 4 to 6, the differentially methylated regions of CASZ1, C1QL3, and PRRX1 genes have a higher sensitivity (88.5%) for CRC and a higher specificity (96.6%) for non-colon cancer samples when validated by the methylated DNA immunoprecipitation enrichment method qPCR detection platform, with an accuracy of 92.4%. The overall performance is better than that of bisulfite conversion treatment qPCR detection.

[0095] Analysis of clinical colorectal cancer staging sample test results Among the 61 clinically diagnosed plasma samples, there were 11 samples of CRC stage 0~I, 12 samples of CRC stage II, 18 samples of CRC stage III, and 20 samples of CRC stage IV. The detection and statistical analysis of samples of different CRC pathological stages based on qPCR detection based on methylated DNA immunoprecipitation enrichment method and bisulfite conversion treatment are shown in Tables 7 and 8: Table 7 Detection of samples in different CRC pathological stages

[0096] Table 8 Statistical analysis of sensitivity of different CRC pathological stages

[0097] As shown in Tables 7 and 8, the differentially methylated regions of CASZ1, C1QL3, and PRRX1 genes also maintained a high sensitivity (90.9% and 83.3%) for CRC stage 0-I and stage II samples when the qPCR detection platform based on the methylated DNA immunoprecipitation enrichment method was verified. The performance for early detection of CRC was better than that of bisulfite conversion treatment qPCR detection, providing a new potential marker for the early detection of colorectal cancer.

[0098] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teaching content of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of a detection reagent for the methylation level of a gene marker combination in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer, characterized in that: The gene marker combination includes CASZ1, C1QL3 and PRRX1.

2. The use according to claim 1, characterized in that: The detection reagents include methylation region enrichment or methylation treatment reagents, and also include qPCR detection reagents.

3. The use according to claim 2, characterized in that: The methylated region enrichment reagent includes a methylated DNA antibody.

4. The use according to claim 3, characterized in that: The qPCR detection reagents include a primer pair and a probe targeting the methylated region.

5. The use according to claim 4, characterized in that: The methylated region of the CASZ1 gene is chr1:10647750-10648000, the primer pair is shown in SEQ ID No. 1 and SEQ ID No. 2, and the probe is shown in SEQ ID No. 3; the methylated region of the C1QL3 gene is chr10:16520568-16520767, the primer pair is shown in SEQ ID No. 4 and SEQ ID No. 5, and the probe is shown in SEQ ID No. 6; the methylated region of the PRRX1 gene is chr1:170661257-170661457, the primer pair is shown in SEQ ID No. 7 and SEQ ID No. 8, and the probe is shown in SEQ ID No.

9.

6. The use according to claim 2, characterized in that: The methylation treatment reagent includes bisulfite.

7. The use according to claim 6, characterized in that: The qPCR detection reagent comprises a primer pair and a probe targeting the methylated region after treatment.

8. The use according to claim 7, characterized in that: The methylated region of the CASZ1 gene is chr1:10647750-10648000, the primer pair is shown in SEQ ID No. 10 and SEQ ID No. 11, and the probe is shown in SEQ ID No. 12; the methylated region of the C1QL3 gene is chr10:16520568-16520767, the primer pair is shown in SEQ ID No. 13 and SEQ ID No. 14, and the probe is shown in SEQ ID No. 15; the methylated region of the PRRX1 gene is chr1:170661257-170661457, the primer pair is shown in SEQ ID No. 16 and SEQ ID No. 17, and the probe is shown in SEQ ID No.

18.

9. Use of a detection reagent for the methylation level of a combination of gene markers in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on the following method: S1, obtain cfDNA samples from biological samples of subjects; S2, enriching methylated regions of cfDNA samples or performing methylation treatment; S3, using the product enriched or treated in step S2 as a template, and using primer pairs and probes targeting the methylated regions of the untreated or treated gene marker combination to perform qPCR amplification; If there is a typical amplification curve and the Ct value is not greater than the preset threshold, the test subjects are diagnosed with colorectal cancer or have a risk of colorectal cancer. in, The gene marker combination includes CASZ1, C1QL3 and PRRX1, the methylation region of the CASZ1 gene is chr1:10647750-10648000; the methylation region of the C1QL3 gene is chr10:16520568-16520767; and the methylation region of the PRRX1 gene is chr1:170661257-170661457.

10. A kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on DNA immunoprecipitation detection of gene marker combination methylation levels, characterized in that: The gene marker combination includes CASZ1, C1QL3 and PRRX1, the methylation region of the CASZ1 gene is chr1:10647750-10648000, the primer pair is shown in SEQ ID No. 10 and SEQ ID No. 11, and the probe is shown in SEQ ID No. 12; the methylation region of the C1QL3 gene is chr10:16520568-16520767, the primer pair is shown in SEQ ID No. 13 and SEQ ID No. 14, and the probe is shown in SEQ ID No. 15; the methylation region of the PRRX1 gene is chr1:170661257-170661457, the primer pair is shown in SEQ ID No. 16 and SEQ ID No. 17, and the probe is shown in SEQ ID No. 18.

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