Methylation marker combination for colorectal cancer diagnosis or prediction, application, kit and computer readable storage medium

Abstract

The invention discloses a methylation molecular marker combination for diagnosing or predicting colorectal cancer, which is composed of the following two methylation molecular markers which are respectively positioned on human SLIT2 and PAX5 genes: chr4: 20253208-20253408 and chr9: 37002603-37002803, and the methylation molecular markers are specifically positioned through hg38. The invention also discloses application of the methylation molecular marker combination, a related kit and an analysis processing program recorded by a computer readable storage medium. The methylation level of the SLIT2 and PAX5 genes is used as a marker for early diagnosis or prediction of the colorectal cancer, and the choices of technicians in the field are enriched.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and genetic testing, and specifically relates to a methylation marker combination and application, a kit and a computer-readable storage medium for diagnosing or predicting colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is a common 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.

[0003] According to the multi-stage theory of carcinogenesis, CRC progression manifests morphologically as a phased progression from normal mucosal hyperplasia to polyp adenoma formation, adenoma carcinomatization, and finally, invasion and metastasis. The progression from polyp adenoma to CRC takes 10-15 years. Early detection of CRC has a cure rate exceeding 90%, while late detection has a cure rate of less than 10%. Therefore, risk identification, screening, and early diagnosis of CRC precancerous lesions are essential to reducing CRC mortality.

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

[0005] Colonoscopy is the gold standard for colorectal cancer screening, but because of its high invasiveness and cumbersome intestinal preparation, the compliance of Chinese people with colonoscopy screening is relatively low. In addition, there is a large demand for colonoscopy examinations. 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 examinations 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 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. In addition, this screening model has resulted in insufficient manpower investment in the hospital, resulting in slow project progress.

[0006] Peripheral blood is one of the most studied types of biological samples. For people who are asymptomatic, at medium risk, and unwilling to undergo stool tests or endoscopy, blood testing may be a popular choice. Based on blood DNA testing, auxiliary diagnosis technology for colorectal cancer, the current marketed products generally have low sensitivity, less than 85%, which cannot meet clinical needs. Clinical practice shows that the main limitation of blood Septin9 methylation testing is its relatively low sensitivity for identifying colorectal cancer and precancerous lesions (adenomas). The sensitivity for advanced adenomas is only 7.9% to 38.7%.

[0007] Colorectal cancer screening technology based on fecal DNA testing mainly targets characteristics such as gene mutations and / or methylation in colorectal exfoliated cells, overcoming the main drawback of detecting microbleeding. It has single-target and multi-target solutions and can also be combined with FIT for testing. It has the advantages of not requiring special equipment, not requiring dietary restrictions, and being non-invasive. Although there have been significant improvements in sensitivity and specificity for colorectal cancer, the detection rate for early stage cancers of grades 0-II is still below 90%, and the detection rate for advanced adenomas such as precancerous lesions and high-grade intraepithelial neoplasia is even lower, less than 65% or even lower. Although fecal DNA testing can be done by sampling at home, stool sampling is relatively private and inconvenient, and is different from the public's medical habits. It will take a long time for the public to accept fecal DNA testing, so there are still certain issues of test compliance in the screening process.

[0008] Therefore, developing new and more sensitive colorectal cancer markers based on blood samples with the highest clinical user compliance has become an urgent issue to be addressed.

[0009] 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 CpG doublet positions. CpG doublets are unevenly distributed in the human genome, and areas of concentrated enrichment 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. Hypermethylation includes but is not limited to tumor suppressor genes, and hypomethylation includes but is not limited to proto-oncogenes.

[0010] All aspects of tumor development and progression may be linked to changes in DNA methylation, including cell cycle regulation, DNA damage repair, biochemical metabolism of carcinogenic compounds, apoptosis, and angiogenesis. Different tumor types may have specific hypermethylated tumor suppressor genes and hypomethylated oncogenes, creating a specific methylation profile for each cancer type, and this profile can be used to identify the cancer type.

[0011] Methylation enrichment technology is an analytical method used to study methylation modifications on DNA. DNA methylation is a key epigenetic modification that involves the addition of methyl groups to the cytosine ring of DNA molecules. This modification plays a key role in biological processes such as gene expression, cell differentiation, and genome stability. Therefore, understanding the status of DNA methylation is crucial for understanding biological processes and the development of diseases.

[0012] Common methylation enrichment techniques include methylation-specific PCR (MSP), methylation-sensitive restriction enzyme digestion, methylated DNA immunoprecipitation (MeDIP), methylated DNA immunoprecipitation sequencing (MeDIP-Seq), and MBD-Seq (Methyl-CpGBinding Domain sequencing), among which:

[0013] 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 genome-wide methylation information and is only applicable to pre-determined target regions.

[0014] Methylation-sensitive restriction enzyme cleavage exploits the differences in restriction enzyme sensitivity 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 in the cytosine ring to restriction enzymes. This technology does not require the use of expensive sequencing technology and can be analyzed through methods such as gel electrophoresis. However, it cannot provide high-resolution information on individual CpG sites and generally only provides information on the methylation status of the entire region. Furthermore, due to the specificity of the selected restriction enzyme, some methylated sites may be missed or over-detected. Furthermore, it cannot directly distinguish between 5-methylcytosine and other forms of DNA modification.

[0015] MeDIP-Seq uses methylated DNA antibodies to selectively enrich methylated DNA fragments, which are then analyzed by high-throughput sequencing. It can enrich the entire methylated genomic region and is suitable for genome-wide methylation analysis. However, it cannot provide high-resolution information on individual CpG sites.

[0016] MBD-Seq uses methylated DNA-binding proteins (such as MBD2 or MBD3) to enrich methylated DNA fragments, which are then analyzed by sequencing. It offers high enrichment efficiency and is suitable for genome-wide methylation analysis. However, similar to MeDIP-Seq, it cannot provide high-resolution information on individual CpG sites.

[0017] Methylation treatment is also known as methylation conversion. Common methylation treatment sequencing technologies include bisulfite sequencing (BS-seq). BS-seq uses bisulfite to treat DNA, converting unmethylated cytosine to uracil while leaving methylated cytosine unaffected. Sequencing analysis is then performed. This technique provides high-resolution information on individual CpG sites and enables genome-wide methylation analysis. However, the experimental procedures are complex.

[0018] The current mainstream method for methylation analysis 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, experiencing the extremes of ice and fire. The resulting converted DNA has the following morphology: mainly single strands, mixed double strands, fragment nicks, gap damage, and uracil nucleotides. This process generally results in the loss of 90% of the DNA template, and a large amount of methylation information cannot be detected by subsequent processes. At the same time, during the base conversion treatment, there are cases of incomplete sequence conversion or over-conversion, which leads to artificial bias, and subsequent PCR amplification will further amplify it, 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 already limited number of free DNA fragments are treated with bisulfite, the difficulty of detecting methylation levels is greatly increased.

[0019] Although the sensitivity for colorectal cancer can be improved by increasing the number of methylated genes based on bisulfite treatment, such as the combined detection of 3 colorectal cancer gene methylation by Beijing Aikelun Medical Technology Co., Ltd., which 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. Summary of the Invention

[0020] One of the technical problems to be solved by the present invention is to provide a methylation molecular marker combination for the diagnosis or prediction of colorectal cancer, which is composed of the following two methylation molecular markers located in the human SLIT2 (Slit Guidance Ligand 2) and PAX5 (Paired Box 5) genes: chr4:20253208-20253408 and chr9:37002603-37002803, which are specifically located by hg38.

[0021] The second technical problem to be solved by the present invention is to provide a reagent for detecting the above-mentioned combination of methylation molecular markers for use in the preparation of products for diagnosing or predicting colorectal cancer.

[0022] In some embodiments, the reagents for detecting the methylation molecular marker combination as described above include reagents used in any one or more of the following methods, including at least one of methylated DNA immunoprecipitation fluorescence quantitative PCR method, bisulfite conversion fluorescence quantitative PCR method, bisulfite conversion sequencing method, methylation chip sequencing, and methylation-specific PCR method.

[0023] In some embodiments, the reagents for detecting the methylation molecular marker combination described above include primer and probe combination a and / or primer and probe combination b;

[0024] Primer and probe combination a: the primer pair shown as SEQ ID No. 1 and SEQ ID No. 2 and the Taqman MGB probe shown as SEQ ID No. 3, used for fluorescent quantitative PCR amplification of chr4: 20253208-20253408; the primer pair shown as SEQ ID No. 4 and SEQ ID No. 5 and the Taqman MGB probe shown as SEQ ID No. 6, used for fluorescent quantitative PCR amplification of chr9: 37002603-37002803;

[0025] Primer and probe combination b: the primer pair shown as SEQ ID No.7 and SEQ ID No.8 and the Taqman MGB probe shown as SEQ ID No.9, used for fluorescent quantitative PCR amplification of chr4:20253208-20253408; the primer pair shown as SEQ ID No.10 and SEQ ID No.11 and the Taqman MGB probe shown as SEQ ID No.12, used for fluorescent quantitative PCR amplification of chr9:37002603-37002803.

[0026] The third technical problem to be solved by the present invention is to provide a kit for diagnosing or predicting colorectal cancer. The kit is used to detect a combination of methylation molecular markers; the combination of methylation molecular markers is composed of the following two methylation molecular markers located in the human SLIT2 and PAX5 genes, respectively: chr4:20253208-20253408 and chr9:37002603-37002803, specifically located using hg38;

[0027] The kit includes a primer and probe combination a and / or a primer and probe combination b;

[0028] Primer and probe combination a: the primer pair shown as SEQ ID No. 1 and SEQ ID No. 2 and the Taqman MGB probe shown as SEQ ID No. 3, used for fluorescent quantitative PCR amplification of chr4: 20253208-20253408; the primer pair shown as SEQ ID No. 4 and SEQ ID No. 5 and the Taqman MGB probe shown as SEQ ID No. 6, used for fluorescent quantitative PCR amplification of chr9: 37002603-37002803;

[0029] Primer and probe combination b: the primer pair shown as SEQ ID No.7 and SEQ ID No.8 and the Taqman MGB probe shown as SEQ ID No.9, used for fluorescent quantitative PCR amplification of chr4:20253208-20253408; the primer pair shown as SEQ ID No.10 and SEQ ID No.11 and the Taqman MGB probe shown as SEQ ID No.12, used for fluorescent quantitative PCR amplification of chr9:37002603-37002803.

[0030] In some embodiments, methylated DNA immunoprecipitation (MeDIP) technology is used to enrich methylated fragments, and then the enriched DNA is used as a template to perform fluorescent quantitative PCR under the conditions of primer and probe combination a; the kit also includes a methylated DNA antibody, which 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-carboxylcytosine (5-caC) antibody.

[0031] In some embodiments, the method further comprises one or more of Rapid Taq Master Mix, reagents required for methylation enrichment based on the principle of 5-methylcytosine antibody, and reagents required for methylation conversion based on the principle of bisulfite conversion.

[0032] In some embodiments, the 3' end of the Taqman MGB probe carries MGB and a fluorescence quencher group, and the 5' end carries a fluorescent group; the combination formed by the fluorescence quencher group and the fluorescent group is selected from BHQ1 or NFQ and FAM, BHQ2 and VIC or HEX, BHQ2 and Cy3, and BHQ2 and Cy5.

[0033] In some embodiments, the two Taqman MGB probes in primer and probe combination a carry different fluorescent quenching groups and fluorescent group combinations, respectively, so that the corresponding Ct values ​​can be read according to the fluorescence color difference in the fluorescent quantitative PCR amplification reaction with the two Taqman MGB probes.

[0034] In some embodiments, the two Taqman MGB probes in primer and probe combination b carry different fluorescence quenching groups and fluorescent group combinations, respectively, so that the corresponding Ct values ​​can be read according to the fluorescence color difference in the fluorescence quantitative PCR amplification reaction with the two Taqman MGB probes.

[0035] In some embodiments, the two Taqman MGB probes of primer and probe combination a carry the same fluorescent quencher and fluorescent group combination.

[0036] In some embodiments, the two Taqman MGB probes of primer and probe combination b carry the same combination of fluorescent quencher and fluorescent group.

[0037] A fourth technical problem to be solved by the present invention is to provide a computer-readable storage medium comprising a program, wherein the program can be executed by a processor to analyze and process the fluorescence quantitative PCR detection data of the methylation molecular marker combination described above to obtain a colorectal cancer determination result, comprising the following steps:

[0038] The Ct values ​​were obtained by fluorescence quantitative PCR using the DNA enriched by cfDNA immunoprecipitation with methylated DNA as template under the conditions of primer and probe combination a. SLIT2 , Ct PAX5 , use formula I and formula II to process and judge the test results;

[0039] Formula I: logistic scores=e k / (1+e k )

[0040] Formula II: k = -2.478 × Ct SLIT2 -3.076×Ct PAX5 +203.157

[0041] A logistic score of ≤800 was considered negative for colorectal cancer, and a logistic score of >800 was considered positive for colorectal cancer;

[0042] If the Ct value obtained by fluorescent quantitative PCR test is greater than 45 or no Ct value is detected, the Ct value is counted as 45 for substitution into Formula II;

[0043] Primer and probe combination a: primer pair as shown in SEQ ID No.1 and SEQ ID No.2 and Taqman MGB probe as shown in SEQ ID No.3, used for fluorescence quantitative PCR amplification of chr4:20253208-20253408 to obtain Ct SLIT2 The primer pairs shown in SEQ ID No.4 and SEQ ID No.5 and the Taqman MGB probe shown in SEQ ID No.6 were used for fluorescence quantitative PCR amplification of chr9:37002603-37002803 to obtain Ct PAX5 .

[0044] The present invention also provides another computer-readable storage medium, comprising a program, wherein the program can be executed by a processor to analyze and process the fluorescence quantitative PCR detection data of the methylation molecular marker combination described above to obtain a colorectal cancer determination result, comprising the following steps:

[0045] The Ct values ​​obtained by fluorescence quantitative PCR using bisulfite-converted DNA as template and primer and probe combination b were SLIT2 , Ct PAX5 , use formula I and formula III to process and judge the test results;

[0046] Formula I: logistic scores=e k / (1+e k )

[0047] Formula III: k = -0.36 × Ct SLIT2 -1.373×Ct PAX5 +67.241

[0048] A logistic score of ≤725 was considered negative for colorectal cancer, and a logistic score of >725 was considered positive for colorectal cancer;

[0049] If the Ct value obtained by fluorescent quantitative PCR test is greater than 45 or no Ct value is detected, the Ct value is counted as 45 for substitution into Formula III;

[0050] Primer and probe combination b: primer pair as shown in SEQ ID No.7 and SEQ ID No.8 and Taqman MGB probe as shown in SEQ ID No.9, used for fluorescence quantitative PCR amplification of chr4:20253208-20253408 to obtain Ct SLIT2 The primer pairs shown in SEQ ID No.10 and SEQ ID No.11 and the Taqman MGB probe shown in SEQ ID No.12 were used for fluorescence quantitative PCR amplification of chr9:37002603-37002803 to obtain Ct PAX5 .

[0051] In some embodiments, the fluorescence quantitative PCR detection data analyzed and processed by the program of the computer-readable storage medium comes from the following fluorescence quantitative PCR reaction: the reaction system is 35 μL; the reaction liquid reagent used is 2×Rapid TaqMaster Mix; the reaction conditions are 95°C for 5 minutes, 95°C for 15 seconds, 60°C for 40 seconds, and 45 cycles of amplification.

[0052] Compared with the prior art, the present invention has the following technical effects:

[0053] The present invention provides methylation regions of the SLIT2 and PAX5 genes (chr4:20253208-20253408 and chr9:37002603-37002803, specifically located by hg38) as methylation molecular markers for diagnosis (including early diagnosis, auxiliary diagnosis) or prediction of colorectal cancer, enriching the options of those skilled in the art.

[0054] The present invention uses methylated DNA immunoprecipitation technology combined with fluorescent quantitative PCR to detect the methylation levels of the methylated regions of the SLIT2 and PAX5 genes, with high sensitivity and strong specificity, and has very important clinical application value.

[0055] The present invention is based on the methylation levels of specific regions of the SLIT2 and PAX5 genes, can achieve early detection of colorectal cancer, and provide more biomarker options (methylation molecular markers are one type of biomarker) for colorectal cancer screening and early diagnosis.

[0056] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0058] Figure 1 The SLIT2 and PAX5 methylation gene detection process in Example 2 of the present invention is shown.

[0059] Figure 2A-2D The amplification curve of a sample based on methylated DNA immunoprecipitation enrichment method qPCR detection is shown ( Figure 2A and 2B ) and the amplification curve of qPCR detection based on bisulfite conversion treatment (as shown in Figure 2C and 2D shown).

[0060] Figure 3A The ROC curve of 122 samples based on methylated DNA immunoprecipitation enrichment method qPCR detection is shown.

[0061] Figure 3B The ROC curve of the qPCR assay based on bisulfite conversion treatment for 122 samples is shown. DETAILED DESCRIPTION

[0062] In order to facilitate understanding by those skilled in the art, some terms appearing in this document are explained and illustrated.

[0063] As used herein, the singular forms "a," "an," and "the" include plural forms unless the context indicates otherwise. Thus, for example, reference to "an agent" is intended to include a plurality of agent components.

[0064] Herein, unless otherwise stated, the terms “comprises”, “includes” or “comprising” mean that the listed values, steps or components are included, but other values, steps or components are not excluded.

[0065] As used herein, "subject" or "patient" are used interchangeably and refer to a vertebrate, preferably a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples.

[0066] Numerical ranges in this application are approximate values, so unless otherwise stated, they may include numerical values ​​outside the scope. Numerical ranges include all numerical values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range comprising a numerical value less than 1 or comprising a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately considered to be 0.0001, 0.001, 0.01 or 0.1. For a range comprising a single digit less than 10 (e.g., 1 to 5), 1 unit is typically considered to be 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of the numerical values ​​between the minimum and maximum values ​​listed are considered to be clearly recorded in this application.

[0067] The term "AUC" is an abbreviation for "area under the curve". It specifically refers to the area under the receiver operating characteristic (ROC) curve. The ROC curve is a plot of the true positive rate relative to the false positive rate for different possible cut-off points of a diagnostic test. It shows the balance between sensitivity and specificity according to the selected cut-off point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under the ROC curve (AUC) is a measure of a diagnostic test (the larger the area, the better; the best is 1; a randomized trial will have an ROC curve with an area on the diagonal of 0.5; Reference: JPEgan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0068] To detect changes in colorectal cancer-specific gene methylation levels in ctDNA, identify new, more sensitive and specific colorectal cancer methylation markers, and establish early screening and diagnosis models, the inventors of this invention have devoted significant effort to developing effective colorectal cancer methylation markers in cfDNA, enabling early diagnosis of cancer and the risk of cancerous transformation. Unexpectedly, they discovered that specific regions of the SLIT2 and PAX5 genes are methylated in colorectal cancer cells. This invention utilizes these specific gene regions as methylation markers.

[0069] In the present invention, after long-term exploration and verification of a large number of clinical samples, the inventors unexpectedly discovered that the methylation levels of the SLIT2 and PAX5 genes are significantly different in colorectal cancer and non-colorectal cancer. SLIT2 is a member of the axon guidance factor ligand family. Its physiological functions are numerous and complex, and it plays an indispensable role in biological development, neurogenesis, tumor progression, and other processes; the PAX5 gene encodes a member of the paired box (PAX) family of transcription factors. The core feature of this gene family is a novel, highly conserved DNA binding motif, the paired box. Paired box transcription factors are important regulatory factors in early development, and changes in their gene expression are believed to contribute to tumor transformation. This gene encodes a B cell lineage-specific activating protein that is expressed early rather than late in B cell differentiation.

[0070] An amplification curve is a graph that shows the accumulation of products 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:

[0071] Initial stage:

[0072] Threshold Cycle (Ct) value: In the early stages of a PCR reaction, the fluorescence signal may be low, but as 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 predetermined threshold. A lower Ct value indicates a higher starting amount of target DNA in the sample.

[0073] Exponential growth phase:

[0074] Exponential phase: During the middle phase of the PCR reaction, the PCR product grows exponentially. The Ct value increases faster, reflecting the exponential growth of the target DNA in the PCR reaction.

[0075] Platform stage:

[0076] Plateau phase: In the later stages of the PCR reaction, the accumulation of PCR products reaches saturation and no longer grows exponentially. The PCR amplification curve at this stage forms a plateau, and the increase in Ct value becomes slow.

[0077] In some embodiments of the present invention, the diagnosis is an early stage diagnosis, specifically, the early stage is colorectal cancer (CRC) stage 0 to stage I or stage II.

[0078] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0079] Example 1 Discovery of Colorectal Cancer-Specific Methylated Gene Sites

[0080] In order to screen for biomarkers specifically methylated in colorectal cancer, this example collected blood samples from 548 patients clinically diagnosed with colorectal cancer (also referred to as colorectal cancer-positive samples) and 389 patients clinically diagnosed with colorectal cancer-negative blood samples.

[0081] 1. Prepare methylated DNA sample library

[0082] (1) DNA extraction

[0083] cfDNA was extracted using a commercial extraction kit according to the instructions.

[0084] Nucleic acid concentration and fragment distribution were quality controlled using Qubit 4.0 and Qsep 100, respectively. The yield of cfDNA extracted from 2 mL of human plasma should be greater than 5 ng, with an enrichment peak at or near 167 bp. When the yield exceeds 50 ng, fragmentation quality control was performed using Qsep 100 capillary electrophoresis. If contamination with large fragments is present, magnetic beads were used for fragment screening to remove the large fragments.

[0085] (2) Library construction

[0086] Commercial library construction kits can be used according to the instructions in the manufacturer's instructions. For example, the Rapid Plus DNA LibPrep Kit for illumina (Cat. No. RK20208, ABclonal) or the VAHTS Universal ProDNA Library Prep Kit for illuminaVazyme (Cat. No. ND608-02, Novazom) or similar kits can be used for end repair, A-tailing, and ligation with adapters. In this example, the VAHTS Universal ProDNA Library Prep Kit for illuminaVazyme (Cat. No. ND608-02, Novazom) was used for library construction.

[0087] (3) Methylated DNA immunoprecipitation

[0088] Each methylation enrichment reaction can simultaneously complete the mixing of 12 to 100 cfDNA libraries (the input amount of each cfDNA sample library is about 10 ng), and can simultaneously complete the mixing of 10 to 24 genomic DNA libraries (the input amount of each genomic DNA library is about 100 ng). The methylation enrichment based on the principle of 5-methylcytosine (5mC) antibody 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 (item number D5101-A). The methylation enrichment reaction is then purified according to the instructions, 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 500 ng per reaction.

[0089] 2. Prepare DNA Probe Library

[0090] (1) Probe design

[0091] The inventors selected the longest SLIT2 and PAX5 gene transcripts from the NCBI database to confirm the gene locations, and obtained sequence information of their promoter region, 5' UTR, first exon (exon 1), and 1 kb upstream of the start codon.

[0092] The obtained target methylation regions of SLIT2 and PAX5 genes were used to design probes. The design principles of the probes are: (1) full coverage of the target region without gaps;

[0093] (2) No overlap;

[0094] (3) Each probe is 120 nt in length.

[0095] The specific genomic locations of the targeted regions and the specific genomic locations of the probe coverage regions designed thereby are detailed in Table 1.

[0096] (2) Probe synthesis

[0097] Using the above probe design principles, a total of 50 probes were designed for the methylation regions of the SLIT2 and PAX5 genes, covering all possible CpG sites. The probe coverage information is shown in Table 1.

[0098] Table 1. Probe coverage information

[0099]

[0100]

[0101] 3. DNA Capture Probe Hybridization

[0102] NadPrep hybridization capture reagent (Cat. No. REF1005101, Naonda) was used for liquid phase hybridization capture. The hybridization capture reaction can be single hybrid or multi-hybrid. The total amount of MeDIP amplified library input for each hybridization 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 Nad Nano Blockers were placed in a vacuum concentrator preheated to 42°C and dried at 1000 rpm. After drying, the prepared hybridization reaction solution (containing the above-mentioned probe panel) was added, and the mixture was vortexed and centrifuged instantaneously. 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 this period, the beads were vortexed every 10 minutes to ensure that the 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.

[0103] 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 at each step; finally, 20 μL of nuclease-free water is added and gently vortexed to mix.

[0104] 4. PCR amplification and purification after hybridization capture

[0105] The hybrid capture product was amplified by PCR using the amplification reagents in the VAHTS Universal Pro DNA Library Prep Kit for illumina (Cat. No. ND608-02, Vazyme) for 12-13 cycles. After amplification, the product was purified using an equal volume of VAHTS DNA Clean Beads (Cat. No. N411-03, Vazyme) to obtain a relatively pure hybrid capture library. Library concentration was quantified using Qubit 4.0, and fragment size was determined using the Qsep 100 fully automated nucleic acid and protein analyzer.

[0106] 5. Library sequencing and bioinformatics analysis

[0107] Dilute the library concentration to be loaded onto the machine to 4 nM and mix according to the required data volume. The total data volume should not exceed 120G. After mixing, take out 5 μL of the library, add 5 μL of 0.2N NaOH, mix by pipetting, and denature for 5 minutes. Immediately after the end, add 990 μL of HT1 Buffer (REF: 15058251, Illumina), vortex to mix, take out 105 μL and add 1295 μL of HT1 Buffer. After vortex mixing, this is the library for loading onto the machine, with a concentration of 1.5 pM.

[0108] The sequencer was an Illumina NextSeq 550Dx, and the reagents used included 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 was added to the sample position of the High Output Reagent Cartridge v2, and each reagent was added in turn to begin sequencing. This example used paired-end sequencing, which took a total of approximately 30 hours.

[0109] 6. Quality Control of Sequencing Data

[0110] Fastp (version 0.22.0) was used to quality control the data and remove low-quality bases. The overall Q20 of the clean data was above 90%, and the Q30 was above 85%. The average sequencing depth was approximately 300×. The average on-target rate of the probes in the above probe combination was above 80%, demonstrating that this example, based on the combination of methylation immunoprecipitation and liquid-phase hybridization capture probes, is feasible and effective for detecting cancer-related methylation regions.

[0111] 7. Analysis of differential methylation regions of SLIT2 and PAX5 genes related to colorectal cancer

[0112] The DiffBind tool (version 3.8.4) was used to screen for differential peaks between tumor and non-tumor groups. Using the DESeq and EdgeR algorithms, an intersection was used to prioritize regions within the panel. The screening criteria were: 1) False Discovery Rate (FDR) < 0.01, 2) Fold Change < -1. The most significantly differentially methylated regions between the colorectal cancer-positive and colorectal cancer-negative groups that met these criteria were identified as differentially methylated regions.

[0113] In this example, from the regions of the SLIT2 and PAX5 genes covered by 50 probes, the characteristic methylation regions with the most significant differences between the colorectal cancer positive group and the colorectal cancer negative group were screened as differentially methylated regions. The methylated CpG sites in these differentially methylated regions are shown in Table 2.

[0114] Table 2. Methylated CpG sites in differentially methylated regions

[0115]

[0116]

[0117] The RPM index (Reads per million mapped reads) of the differentially methylated regions (covering the reads region of the CpG sites in Table 2) in 548 colorectal cancer-positive samples and 389 colorectal cancer-negative samples was analyzed. The P value was less than 0.005, indicating a significant difference.

[0118] Example 2 SLIT2 and PAX5 methylation gene detection in clinical samples and comparison of different treatment schemes

[0119] To further validate the clinical performance of the differentially methylated regions of the colorectal cancer-related SLIT2 and PAX5 genes in colorectal cancer plasma samples, the inventors used qPCR to test two additional groups of samples (62 plasma samples clinically diagnosed with CRC and 60 plasma control samples negative for CRC by colonoscopy). Among the 62 plasma samples clinically diagnosed with CRC, 14 samples were from CRC stages 0-1, 15 samples were from CRC stage II, 15 samples were from CRC stage III, and 18 samples were from CRC stage IV.

[0120] SLIT2 and PAX5 methylation gene detection flow chart as follows Figure 1 As shown, specifically:

[0121] (1) DNA extraction

[0122] cfDNA was extracted using a commercial extraction kit according to the instructions.

[0123] Nucleic acid concentration and fragment distribution quality control are performed using Qubit 4.0 and Qsep 100, respectively. The yield of cfDNA extracted from 4 mL of human plasma should be greater than 10 ng, with an enrichment peak at or near 167 bp. If the yield exceeds 50 ng, fragmentation quality control should be performed using Qsep 100 capillary electrophoresis. If contamination with large fragments is present, fragment screening with magnetic beads should be performed to remove the large fragments.

[0124] (2) Methylated DNA treatment

[0125] ① Methylated DNA immunoprecipitation

[0126] Half of the total amount of nucleic acid extracted above was used for cfDNA methylation enrichment, which was carried out in different reactions. The methylation enrichment based on the 5mC antibody principle was performed using the zymoMeDIP kit (Cat. No. D5101-A). The methylation enrichment reaction was followed by purification according to the instructions, and the elution volume was 50 μL.

[0127] ② Bisulfite conversion of methylated DNA

[0128] Half of the total amount of nucleic acid extracted above was subjected to bisulfite treatment for cfDNA methylation. This was performed in separate reactions. Methylated DNA treatment based on the bisulfite conversion principle was performed using the ZYMO RESEARCH Biotechnology Company DNA Methylation Kit (EZ DNA Methylation Kit, D5002). The elution volume was 50 μL.

[0129] (3) qPCR detection

[0130] The primers and probes were synthesized at Shanghai Bio-Tech Co., Ltd. The specific sequence information is as follows:

[0131] The sequences of the Taqman MGB probe primer pairs for methylated DNA immunoprecipitation enrichment are shown in Table 3.

[0132] Table 3. Primer pairs for Taqman MGB probes for methylated DNA immunoprecipitation enrichment

[0133]

[0134] The probe was labeled with MGB at the 3' end, a fluorescent group (FAM) at the 5' end, and a quencher group (BHQ1) at the 3' end.

[0135] PCR amplification was performed using methylated DNA enriched by immunoprecipitation as a template. The final concentration of each primer was 10 μM, and each gene was amplified using a single-plex reaction. The PCR reaction system consisted of 5 μL of enriched template DNA, 2.5 μL of the aforementioned primer premix, and 17.5 μL of 2× Rapid Taq Master Mix, with the total volume brought to 35 μL. PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 15 seconds, and 60°C for 40 seconds, for 45 cycles.

[0136] The sequences of the Taqman MGB probe primer pairs after bisulfite conversion are shown in Table 4.

[0137] Table 4. Taqman MGB probe primer pairs after bisulfite conversion

[0138]

[0139] The probe was labeled with MGB at the 3' end, a fluorescent group (FAM) at the 5' end, and a quencher group (BHQ1) at the 3' end.

[0140] PCR amplification was performed using bisulfite-converted DNA as a template. The final concentration of each primer was 10 μM, and each gene was amplified in a single-plex reaction. The PCR reaction system consisted of 5 μL of enriched template DNA, 2.5 μL of the aforementioned primer premix, and 17.5 μL of 2× Rapid Taq Master Mix, with the total volume brought to 35 μL. PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 15 seconds, and 60°C for 40 seconds, for 45 cycles.

[0141] (4) Analysis of clinical sample test results

[0142] The offline data were analyzed, and 122 samples were enriched by methylated DNA immunoprecipitation method and bisulfite treatment DNA method, and then detected by qPCR.

[0143] Figure 2A-2D The following are examples of qPCR amplification curves for samples from the same CRC patient (sample number: CRC104435) using the methylated DNA immunoprecipitation enrichment method and the bisulfite treatment DNA method. Figure 2A and 2B These are the qPCR amplification curves of SLIT2 and PAX5 genes after immunoprecipitation enrichment of methylated DNA, with Ct values ​​of 33.14 and 35.98, respectively. Figure 2C and 2D The qPCR amplification curve of bisulfite-treated DNA was obtained, with Ct values ​​of 38.23 and 38.03. The qPCR detection after immunoprecipitation enrichment of methylated DNA had obvious advantages.

[0144] The Ct value of samples with a Ct value of >45 or no Ct value (Undetermined) was set to 45, and the logistic regression formula was used to calculate and draw ROC curves according to the calculation results. Figure 3A and Figure 3B As shown, the area under the curve (AUC) of the ROC curves obtained based on the two different methods were 0.969 and 0.958, respectively. According to the ROC curves, thresholds (cut-off values) were set for different methods: based on the methylated DNA immunoprecipitation enrichment method qPCR detection (i.e., methylated DNA immunoprecipitation fluorescence quantitative PCR method), the cut-off value was set to logistic scores = 800; based on the bisulfite conversion treatment qPCR detection (i.e., bisulfite conversion fluorescence quantitative PCR method), the cut-off value was set to logistic scores = 725. If the logistic scores of the SLIT2 and PAX5 gene amplification of the tested sample are equal to or lower than the set cut-off value, the sample is judged to be negative, otherwise it is judged to be positive. Thus, the test results of 122 samples were statistically analyzed. The formula is as follows:

[0145] Formula I: logistic scores=e k / (1+e k )

[0146] Methylated DNA immunoprecipitation (Formula II): k = -2.478 × Ct SLIT2 -3.076×Ct PAX5 +203.157 Bisulfite conversion (Formula III): k = -0.36 × CtSLIT2 -1.373×Ct PAX5 +67.241

[0147] Table 5 shows a comparison of qPCR detection based on the methylated DNA immunoprecipitation enrichment method and colonoscopy results (gold standard), Table 6 shows a comparison of qPCR detection results based on bisulfite conversion treatment and colonoscopy results, and Table 7 shows a comparison of detection results based on qPCR detection based on the methylated DNA immunoprecipitation enrichment method and qPCR detection based on bisulfite conversion treatment.

[0148] Table 5. Comparison of qPCR results based on methylated DNA immunoprecipitation enrichment method with colonoscopy results

[0149]

[0150] Table 6. qPCR results based on bisulfite conversion compared with colonoscopy results

[0151]

[0152] Table 7. Comparison of enrichment methods based on methylated DNA immunoprecipitation and bisulfite conversion

[0153]

[0154] As shown in Tables 5 to 7, differentially methylated regions of the SLIT2 and PAX5 genes demonstrated a higher sensitivity (95.2%) for CRC and a high specificity (95.0%) for non-colorectal cancer samples when validated using a qPCR detection platform based on methylated DNA immunoprecipitation enrichment. The accuracy reached 95.1%, demonstrating overall superior performance to bisulfite conversion qPCR.

[0155] (5) Analysis of clinical colorectal cancer staging sample test results

[0156] Among the 37 clinically diagnosed CRC plasma samples, 14 were in CRC stages 0-1, 15 were in CRC stage II, 15 were in CRC stage III, and 18 were in CRC stage IV. The detection and statistical analysis of samples with different CRC pathological stages using qPCR detection based on methylated DNA immunoprecipitation enrichment compared with qPCR detection based on bisulfite conversion treatment are shown in Tables 8 and 9.

[0157] Table 8. Detection of samples in different CRC pathological stages

[0158]

[0159] Table 9. Statistical analysis of sensitivity of different CRC pathological stages

[0160]

[0161] As can be seen from Tables 8 and 9, the differentially methylated regions of the SLIT2 and PAX5 genes also maintained a high sensitivity (92.9%, 93.3%) for CRC stage 0-I and stage II samples when validated based on the methylated DNA immunoprecipitation enrichment method qPCR detection platform. The performance for early detection of CRC is better than that of bisulfite conversion treatment qPCR detection, providing a new potential marker for early detection of CRC. Since the present invention maintains a high sensitivity in the early detection of CRC cancer, it can be used for the prediction of CRC. The present invention uses cfDNA as an analysis sample, which is easy to obtain, and because cfDNA contains a lot of information, it is not only limited to the diagnosis (including auxiliary diagnosis) and / or prediction of CRC, but can also be used for the prediction and / or diagnosis of other diseases. It has the characteristics of being superior to single tissue samples in the context of high-throughput sequencing analysis.

[0162] Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. Changes and improvements to the present invention will be possible without exceeding the concept and scope specified in the claims. In summary, the contents of the embodiments of this specification should not be understood as limiting the present invention.

Claims

1. A methylation molecular marker combination for diagnosing or predicting colorectal cancer, characterized in that: It consists of the following two methylation molecular markers located in the human SLIT2 and PAX5 genes, respectively: chr4:20253208-20253408 and chr9:37002603-37002803, and was specifically mapped using hg38.

2. Use of a reagent for detecting the methylation molecular marker combination as claimed in claim 1 in the preparation of a product for diagnosing or predicting colorectal cancer.

3. The use according to claim 2, characterized in that The reagents for detecting the methylation molecular marker combination as described in claim 1 include reagents used in any one or more of the following methods, including at least one of methylated DNA immunoprecipitation fluorescence quantitative PCR method, bisulfite conversion fluorescence quantitative PCR method, bisulfite conversion sequencing method, methylation chip sequencing, and methylation-specific PCR method.

4. The use according to claim 2, characterized in that The reagent for detecting the methylation molecular marker combination according to claim 1 comprises a primer and probe combination a and / or a primer and probe combination b; Primer and probe combination a: the primer pair shown as SEQ ID No. 1 and SEQ ID No. 2 and the Taqman MGB probe shown as SEQ ID No. 3, used for fluorescent quantitative PCR amplification of chr4: 20253208-20253408; the primer pair shown as SEQ ID No. 4 and SEQ ID No. 5 and the Taqman MGB probe shown as SEQ ID No. 6, used for fluorescent quantitative PCR amplification of chr9: 37002603-37002803; Primer and probe combination b: the primer pair shown as SEQ ID No.7 and SEQ ID No.8 and the Taqman MGB probe shown as SEQ ID No.9, used for fluorescent quantitative PCR amplification of chr4:20253208-20253408; the primer pair shown as SEQ ID No.10 and SEQ ID No.11 and the Taqman MGB probe shown as SEQ ID No.12, used for fluorescent quantitative PCR amplification of chr9:37002603-37002803.

5. A kit for diagnosing or predicting colorectal cancer, characterized in that: The kit is used to detect a methylation molecular marker combination; the methylation molecular marker combination consists of the following two methylation molecular markers located in the human SLIT2 and PAX5 genes, respectively: chr4:20253208-20253408 and chr9:37002603-37002803, specifically located using hg38; The kit includes a primer and probe combination a and / or a primer and probe combination b; Primer and probe combination a: the primer pair shown as SEQ ID No. 1 and SEQ ID No. 2 and the Taqman MGB probe shown as SEQ ID No. 3, used for fluorescent quantitative PCR amplification of chr4: 20253208-20253408; the primer pair shown as SEQ ID No. 4 and SEQ ID No. 5 and the Taqman MGB probe shown as SEQ ID No. 6, used for fluorescent quantitative PCR amplification of chr9: 37002603-37002803; Primer and probe combination b: the primer pair shown as SEQ ID No.7 and SEQ ID No.8 and the Taqman MGB probe shown as SEQ ID No.9, used for fluorescent quantitative PCR amplification of chr4:20253208-20253408; the primer pair shown as SEQ ID No.10 and SEQ ID No.11 and the Taqman MGB probe shown as SEQ ID No.12, used for fluorescent quantitative PCR amplification of chr9:37002603-37002803.

6. The kit according to claim 5, wherein It also includes one or more of Rapid Taq Master Mix, reagents required for methylation enrichment based on the principle of 5-methylcytosine antibody, and reagents required for methylation conversion based on the principle of bisulfite conversion.

7. The kit according to claim 5, wherein The 3' end of the Taqman MGB probe carries MGB and a fluorescence quenching group, and the 5' end carries a fluorescent group; the combination formed by the fluorescence quenching group and the fluorescent group is selected from BHQ1 or NFQ and FAM, BHQ2 and VIC or HEX, BHQ2 and Cy3, and BHQ2 and Cy5.

8. A computer-readable storage medium, characterized in that The method comprises a program that can be executed by a processor to analyze and process the fluorescence quantitative PCR detection data of the methylation molecular marker combination according to claim 1 to obtain a colorectal cancer determination result, comprising the following steps: The Ct values ​​were obtained by fluorescence quantitative PCR using the DNA enriched by cfDNA immunoprecipitation with methylated DNA as template under the conditions of primer and probe combination a. SLIT2 , Ct PAX5 , use formula I and formula II to process and judge the test results; Formula I: logistic scores = e k / (1+e k ) Formula II: k = -2.478 × Ct SLIT2 -3.076×Ct PAX5 +203.157 A logistic score of ≤800 was considered negative for colorectal cancer, and a logistic score of >800 was considered positive for colorectal cancer; If the Ct value obtained by fluorescent quantitative PCR test is greater than 45 or no Ct value is detected, the Ct value is counted as 45 for substitution into Formula II; Primer and probe combination a: primer pair as shown in SEQ ID No.1 and SEQ ID No.2 and Taqman MGB probe as shown in SEQ ID No.3, used for fluorescence quantitative PCR amplification of chr4:20253208-20253408 to obtain Ct SLIT2 The primer pairs shown in SEQ ID No.4 and SEQ ID No.5 and the Taqman MGB probe shown in SEQ ID No.6 were used for fluorescence quantitative PCR amplification of chr9:37002603-37002803 to obtain Ct PAX5 .

9. A computer-readable storage medium, characterized in that The method comprises a program that can be executed by a processor to analyze and process the fluorescence quantitative PCR detection data of the methylation molecular marker combination according to claim 1 to obtain a colorectal cancer determination result, comprising the following steps: The Ct values ​​obtained by fluorescence quantitative PCR using bisulfite-converted DNA as template and primer and probe combination b were SLIT2 , Ct PAX5 , use formula I and formula III to process and judge the test results; Formula I: logistic scores = e k / (1+e k ) Formula III: k = -0.36 × Ct SLIT2 -1.373×Ct PAX5 +67.241 A logistic score of ≤725 was considered negative for colorectal cancer, and a logistic score of >725 was considered positive for colorectal cancer; If the Ct value obtained by fluorescent quantitative PCR test is greater than 45 or no Ct value is detected, the Ct value is counted as 45 for substitution into Formula III; Primer and probe combination b: primer pair as shown in SEQ ID No.7 and SEQ ID No.8 and Taqman MGB probe as shown in SEQ ID No.9, used for fluorescence quantitative PCR amplification of chr4:20253208-20253408 to obtain Ct SLIT2 The primer pair shown in SEQ ID No.10 and SEQ ID No.11 and the Taqman MGB probe shown in SEQ ID No.12 were used for fluorescence quantitative PCR amplification of chr9:37002603-37002803 to obtain Ct PAX5 .

10. The computer-readable storage medium according to claim 8 or 9, wherein: The fluorescence quantitative PCR detection data analyzed and processed by the program are from the following fluorescence quantitative PCR reaction: the reaction system is 35 μL; the reaction liquid reagent used is 2× Rapid Taq Master Mix; the reaction conditions are 95°C for 5 minutes, 95°C for 15 seconds, 60°C for 40 seconds, and 45 cycles of amplification.

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