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

Abstract

The invention discloses a methylation molecular marker combination for stomach cancer diagnosis or prediction, which is composed of the following two methylation molecular markers respectively located on human OLIG2 and FERD3L genes: chr21: 33025814-33026014 and chr7: 19145345-19145545, and specific positioning is carried out 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. OLIG2 and FERD3L gene methylation levels are used as markers for early diagnosis or prediction of gastric cancer, and choices of technicians in the field are enriched.

Description

Technical Field

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

[0002] Gastroscopy, the gold standard for gastric cancer diagnosis, is currently widely used in my country. However, due to its invasive nature, relatively high cost, and low public acceptance, it has been difficult to implement for large-scale gastric cancer screening in my country. Furthermore, gastroscopy is primarily performed on symptomatic patients seeking medical attention, and its sensitivity and specificity are suboptimal, resulting in a low rate of early cancer detection.

[0003] Helicobacter pylori (H. pylori) infection is the most significant and most common preventable cause of gastric cancer. On January 6, 2024, Wei Wenqiang and others from the National Cancer Center of the Chinese Academy of Medical Sciences and Peking Union Medical College published a research paper (Trends in the burden of risk factors for gastric cancer in China from 2000 to 2050) in a sub-journal of The Lancet. It is predicted that by 2050, 40.7% of cases of cardia gastric cancer and 62.1% of cases of non-cardia gastric cancer will be caused by Helicobacter pylori infection. Although Helicobacter pylori infection is one of the main causes of gastric cancer, at least 90% of gastric cancers are related to Helicobacter pylori infection, but not all infected people will develop gastric cancer. This means that Helicobacter pylori infection is a risk factor, but not the only determining factor.

[0004] Currently, commonly used gastric cancer screening methods include serum pepsinogen (PG), serum gastrin, and Helicobacter pylori (Hp) antibody testing, as well as barium meal examinations and endoscopy. Existing screening guidelines recommend gastric cancer screening for high-risk individuals aged 40 or 45 years or older. In China, there are over 300 million people at high risk of gastric cancer. Due to economic and medical resource considerations, there is an urgent need to stratify the screening population by risk before gastroscopy to identify high-risk individuals and improve screening efficiency.

[0005] 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. For auxiliary diagnostic technologies for gastric cancer based on blood DNA testing, the current marketed products generally have low sensitivity, all less than 85%, which cannot meet clinical needs. Clinical practice has shown that the main limitation of blood methylation testing is its relatively low sensitivity for identifying gastric cancer, which is 61.76% to 80.77%. Therefore, trying to discover methylation markers of gastric cancer-related genes 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.

[0006] Almost all tumors are caused and promoted by both genetic alterations 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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:

[0011] MSP uses methylation-specific primers to selectively amplify methylated DNA fragments through PCR. It is simple and rapid, suitable for analyzing specific CpG sites, but it cannot provide genome-wide methylation information and is only applicable to pre-defined target regions.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] Methylation treatment is also known as methylation conversion. Common methylation treatment sequencing techniques include bisulfite sequencing (BS-seq). BS-seq uses bisulfite to treat DNA, converting unmethylated cytosine to uracil while leaving methylated cytosine unaffected. This is then analyzed by sequencing. It can provide high-resolution information on individual CpG sites, enabling genome-wide methylation analysis. However, the experimental procedures are cumbersome.

[0016] Research on early cancer screening has found that circulating cell-free DNA (cfDNA) methylation testing, as a non-invasive, low-cost, sensitive, and accurate basis for early tumor detection, can be used for cancer screening and classification of various cancers. Methylation of circulating tumor DNA (ctDNA) is a key epigenetic modification. Gene methylation occurs before cancer and is a key mechanism of cancer development, acting as a "switch" for regulating gene expression. Its stability and consistency make it an ideal tool for early cancer screening. Summary of the Invention

[0017] One of the technical problems to be solved by the present invention is to provide a methylation molecular marker combination for diagnosing or predicting gastric cancer, which is composed of the following two methylation molecular markers located in the human OLIG2 (Oligodendrocyte Transcription Factor 2) and FERD3L (Fer3Like BHLH Transcription Factor) genes: chr21:33025814-33026014 and chr7:19145345-19145545, which are specifically located by hg38.

[0018] The second technical problem to be solved by the present invention is to provide an application of a methylation marker combination for gastric cancer in the preparation of a product, wherein the product is used for diagnosing or predicting gastric cancer; the methylation marker combination for gastric cancer is composed of the following two methylation molecular markers located in the human OLIG2 and FERD3L genes, respectively: chr21:33025814-33026014 and chr7:19145345-19145545, which are specifically located by hg38.

[0019] In some embodiments, the steps include:

[0020] A1, cfDNA samples were enriched by methylated DNA immunoprecipitation;

[0021] A2, using the enriched product of step A1 as a template, perform fluorescence quantitative PCR amplification under primer and probe combination a to obtain Ct OLIG2 and Ct FERD3LPrimer 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 chr21:33025814-33026014 to obtain Ct OLIG2 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 chr7:19145345-19145545 to obtain Ct FERD3L ;

[0022] A3. Process and judge the test results according to Formula I and Formula II;

[0023] Formula I: logistic scores=e k / (1+e k )

[0024] Formula II: k = -2.91 × Ct OLIG2 -1.402×Ct FERD3L +152.908

[0025] A logistic score of ≤650 was considered negative for gastric cancer, and a logistic score of >650 was considered positive for gastric cancer;

[0026] If the Ct value obtained by the fluorescent quantitative PCR test result is greater than 45 or no Ct value is detected, the Ct value is counted as 45 for substitution into Formula II.

[0027] In some embodiments, the steps include:

[0028] B1, cfDNA samples were converted by bisulfite;

[0029] B2, using the conversion product of step B1 as a template, and using primer and probe combination b, perform fluorescence quantitative PCR amplification to obtain Ct OLIG2 and Ct FERD3L Primer and probe combination b: primer pairs 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 chr21:33025814-33026014 to obtain Ct OLIG2The 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 chr7:19145345-19145545 to obtain Ct FERD3L ;

[0030] B3. Process and judge the test results according to Formula I and Formula III;

[0031] Formula I: logistic scores=e k / (1+e k )

[0032] Formula III: k = -1.09 × Ct OLIG2 -0.429×Ct FERD3L +56.945

[0033] A logistic score of ≤650 was considered negative for gastric cancer, and a logistic score of >650 was considered positive for gastric cancer;

[0034] If the Ct value obtained by the fluorescent quantitative PCR test result is greater than 45 or no Ct value is detected, the Ct value is counted as 45 for substitution into Formula III.

[0035] The third technical problem to be solved by the present invention is to provide a kit for diagnosing or predicting gastric cancer. The kit is used to detect the methylation molecular marker combination as described in claim 1;

[0036] The kit includes a primer and probe combination a and / or a primer and probe combination b;

[0037] Primer and probe combination a: the primer pair shown in SEQ ID No. 1 and SEQ ID No. 2 and the Taqman MGB probe shown in SEQ ID No. 3, used for fluorescent quantitative PCR amplification of chr21: 33025814-33026014; the primer pair shown in SEQ ID No. 4 and SEQ ID No. 5 and the Taqman MGB probe shown in SEQ ID No. 6, used for fluorescent quantitative PCR amplification of chr7: 19145345-19145545;

[0038] 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 chr21:33025814-33026014; 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 chr7:19145345-19145545.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] In some embodiments, the two Taqman MGB probes of primer and probe combination a carry the same fluorescent quencher and fluorescent group combination.

[0045] In some embodiments, the two Taqman MGB probes of primer and probe combination b carry the same combination of fluorescent quencher and fluorescent group.

[0046] 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 as described in claim 1 to obtain a gastric cancer determination result, comprising the following steps:

[0047] 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. OLIG2 and Ct FERD3L , use formula I and formula II to process and judge the test results;

[0048] k=-2.91×Ct OLIG2 -1.402×Ct FERD3L +152.908

[0049] A logistic score of ≤650 was considered negative for gastric cancer, and a logistic score of >650 was considered positive for gastric cancer;

[0050] 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;

[0051] 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 chr21:33025814-33026014 to obtain Ct OLIG2 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 chr7:19145345-19145545 to obtain Ct FERD3L .

[0052] The present invention also provides another computer-readable storage medium, comprising a program, wherein the program can be executed by a processor to implement analysis and processing of the fluorescence quantitative PCR detection data of the methylation molecular marker combination according to claim 1 to obtain a gastric cancer determination result, comprising the following steps:

[0053] The Ct values ​​obtained by fluorescence quantitative PCR using bisulfite-converted DNA as template and primer and probe combination b were OLIG2 and Ct FERD3L , use formula I and formula III to process and judge the test results;

[0054] Formula I: logistic scores=e k / (1+e k )

[0055] Formula III: k = -1.09 × Ct OLIG2 -0.429×Ct FERD3L +56.945

[0056] A logistic score of ≤650 was considered negative for gastric cancer, and a logistic score of >650 was considered positive for gastric cancer;

[0057] 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;

[0058] 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 chr21:33025814-33026014 to obtain Ct OLIG2 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 chr7:19145345-19145545 to obtain Ct FERD3L .

[0059] 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.

[0060] Compared with the prior art, the present invention has the following technical effects:

[0061] The present invention provides methylation regions of the OLIG2 and FERD3L genes (chr21: 33025814-33026014, chr7: 19145345-19145545, specifically located by hg38) as methylation molecular markers for the diagnosis (including early diagnosis and auxiliary diagnosis) or prediction of gastric cancer, enriching the options of those skilled in the art.

[0062] The present invention uses methylated DNA immunoprecipitation technology combined with fluorescent quantitative PCR to detect the methylation levels of the methylated regions of the OLIG2 and FERD3L genes, which has high sensitivity and strong specificity and is of great clinical application value.

[0063] The present invention is based on the methylation levels of specific regions of the OLIG2 and FERD3L genes, which can achieve early detection of gastric cancer and provide more biomarker options (methylation molecular markers are one type of biomarker) for gastric cancer screening and early diagnosis.

[0064] 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

[0065] 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.

[0066] Figure 1 The figure shows the process of detecting OLIG2 and FERD3L methylation genes in Example 2 of the present invention.

[0067] Figures 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).

[0068] Figure 3A The ROC curve of 124 samples was shown for qPCR detection based on the methylated DNA immunoprecipitation enrichment method.

[0069] Figure 3BThe ROC curve of the qPCR assay based on bisulfite conversion treatment for 124 samples is shown. DETAILED DESCRIPTION

[0070] In order to facilitate understanding by those skilled in the art, some terms appearing in this document are explained and illustrated.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] As used herein, a "normal healthy" sample refers to a sample of the same type isolated from an individual known to be free of the cancer, tumor, polyp, or adenoma.

[0075] 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).

[0076] To detect gastric cancer-specific changes in methylation levels in ctDNA, identify new gastric cancer methylation markers with higher sensitivity and specificity, and establish early screening and diagnosis models, the inventors of this invention have devoted significant effort to developing effective gastric cancer methylation markers in cfDNA, enabling early diagnosis of cancer and the risk of cancerous transformation. Unexpectedly, they discovered that specific regions of the OLIG2 and FERD3L genes are methylated in gastric cancer cells. This invention utilizes these specific regions of the genes as methylation markers.

[0077] 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 OLIG2 and FERD3L genes are significantly different in gastric cancer and non-gastric cancer. OLIG2 is the main regulator of oligodendrocyte lineage specification, especially regulating the critical stage of early oligodendrocyte development. OLIG2 determines the cell fate of motor neurons, but acts as a gene suppressor. In the process of cell fate conversion, the master regulator reconstructs epigenetic patterns genome-wide, thereby guiding new transcriptomes. FERD3L (Fer3-like BHLH transcription factor) is a gene encoding a protein. Diseases associated with FERD3L include Saethre-Chotzen syndrome and Apert syndrome.

[0078] Early detection of cancers is possible through DNA methylation analysis related to specific cancer types. The current mainstream methylation analysis method involves bisulfite treatment, a process that involves denaturation, deamination, and desulfonation. DNA is first denatured into single strands and then subjected to high temperatures, high salt concentrations, acidic and alkaline environments, experiencing extremes of heat and fire. The resulting converted DNA has a morphology of predominantly single strands, mixed double strands, fragments with nicks, gap damage, and uracil-like nucleotides. This process typically results in the loss of 90% of the DNA template, making a significant amount of methylation information undetectable in subsequent steps. Furthermore, during the base conversion treatment, incomplete or over-conversion of the sequence can occur, leading to artifactual bias. Subsequent PCR amplification can further amplify the sequence and cause inaccurate signals. Therefore, current methylation markers derived from bisulfite treatment generally suffer from low sensitivity, particularly in blood samples, where the already limited number of free DNA fragments becomes significantly more difficult to detect after bisulfite treatment.

[0079] 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:

[0080] Initial stage:

[0081] 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.

[0082] Exponential growth phase:

[0083] 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.

[0084] Platform stage:

[0085] 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.

[0086] In some embodiments of the present invention, the diagnosis is an early diagnosis, specifically, the early stage is stage 0 to stage I or stage II of gastric cancer.

[0087] 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.

[0088] Example 1 Discovery of Gastric Cancer-Specific Methylated Gene Sites

[0089] In order to screen for biomarkers specifically methylated in gastric cancer, this example collected blood samples from 245 patients clinically diagnosed with gastric cancer (also referred to as gastric cancer positive samples) and 306 patients clinically diagnosed with gastric cancer negative.

[0090] 1. Prepare methylated DNA sample library

[0091] (1) DNA extraction

[0092] cfDNA was extracted using a commercial extraction kit according to the instructions.

[0093] 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.

[0094] (2) Library construction

[0095] Commercial library construction kits can be used according to the instructions in the manufacturer's instructions. For example, the Rapid Plus DNALibPrep Kit for illumina (Cat. No. RK20208, ABclonal) or the VAHTS Universal ProDNALibrary Prep Kit for illuminaVazyme (Cat. No. ND608-02, Novaeda) or similar kits can be used for end repair, A-tailing, and ligation with adapters. In this example, the VAHTS Universal Pro DNALibrary Prep Kit for illuminaVazyme (Cat. No. ND608-02, Novaeda) was used for library construction.

[0096] (3) Methylated DNA immunoprecipitation

[0097] 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 10 ng), 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 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 primers to obtain a methylated DNA fragment library with a yield of more than 500 ng per reaction.

[0098] 2. Prepare DNA Probe Library

[0099] (1) Probe design

[0100] The inventors selected the longest OLIG2 and FERD3L 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.

[0101] The probes were designed for the target methylation regions of OLIG2 and FERD3L genes. Principles of probe design:

[0102] (1) Full coverage of the target area without gaps;

[0103] (2) No overlap;

[0104] (3) Each probe is 120 nt in length.

[0105] 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.

[0106] (2) Probe synthesis

[0107] Using the above probe design principles, a total of 62 probes were designed for the methylation regions of the OLIG2 and FERD3L genes, covering all possible CpG sites. The probe coverage information is shown in Table 1.

[0108] Table 1. Probe coverage information

[0109]

[0110]

[0111] 3. DNA Capture Probe Hybridization

[0112] 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.

[0113] 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.

[0114] 4. PCR amplification and purification after hybridization capture

[0115] 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.

[0116] 5. Library sequencing and bioinformatics analysis

[0117] 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.

[0118] 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.

[0119] 6. Quality Control of Sequencing Data

[0120] 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.

[0121] 7. Analysis of differentially methylated regions of gastric cancer-related OLIG2 and FERD3L genes

[0122] 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 employed 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 gastric cancer and non-gastric cancer groups that met these criteria were identified as differentially methylated regions.

[0123] In this example, the OLIG2 and FERD3L gene regions covered by 62 probes were screened for the most significant characteristic methylation regions between the gastric cancer group and the non-gastric cancer group as differentially methylated regions. The methylated CpG sites in these differentially methylated regions are shown in Table 2.

[0124] Table 2. Methylated CpG sites in differentially methylated regions

[0125] Serial number Chromosome location Start site Termination site Serial number Chromosome location Start site Termination site 1 chr21 33025823 33025824 17 chr21 33025957 33025958 2 chr21 33025844 33025845 18 chr21 33025966 33025967 3 chr21 33025858 33025859 19 chr21 33025970 33025971 4 chr21 33025864 33025865 20 chr21 33025998 33025999 5 chr21 33025870 33025871 21 chr21 33026003 33026004 6 chr21 33025876 33025877 21 chr7 19145348 19145349 7 chr21 33025882 33025883 22 chr7 19145357 19145358 8 chr21 33025885 33025886 23 chr7 19145363 19145364 9 chr21 33025888 33025889 24 chr7 19145368 19145369 10 chr21 33025891 33025892 25 chr7 19145389 19145390 11 chr21 33025900 33025901 26 chr7 19145397 19145398 12 chr21 33025914 33025915 27 chr7 19145409 19145410 13 chr21 33025918 33025919 28 chr7 19145463 19145464 14 chr21 33025934 33025935 29 chr7 19145516 19145517 15 chr21 33025952 33025953 30 chr7 19145521 19145522 16 chr21 33025955 33025956 31 chr7 19145542 19145543

[0126] 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 245 gastric cancer-positive samples and 306 gastric cancer-negative samples was analyzed, and the P value was less than 0.005, indicating a significant difference.

[0127] Example 2 OLIG2 and FERD3L methylation gene detection in clinical samples and comparison of different treatment regimens

[0128] To further validate the clinical performance of differentially methylated regions of the gastric cancer-related OLIG2 and FERD3L genes in gastric cancer plasma samples, the inventors used qPCR to test two additional groups of samples (61 plasma samples clinically diagnosed with gastric cancer and 63 plasma control samples with negative gastroscopy results). Among the 61 plasma samples clinically diagnosed with gastric cancer, 16 were from stages 0 to 1, 11 were from stage II, 18 were from stage III, and 16 were from stage IV.

[0129] OLIG2 and FERD3L methylation gene detection flow chart as follows Figure 1 As shown, specifically:

[0130] (1) DNA extraction

[0131] cfDNA was extracted using a commercial extraction kit according to the instructions.

[0132] 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.

[0133] (2) Methylated DNA treatment

[0134] ① Methylated DNA immunoprecipitation

[0135] 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.

[0136] ② Bisulfite conversion of methylated DNA

[0137] 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.

[0138] (3) qPCR detection

[0139] The primers and probes were synthesized at Shanghai Bio-Tech Co., Ltd. The specific sequence information is as follows:

[0140] The sequences of the Taqman MGB probe primer pairs for methylated DNA immunoprecipitation enrichment are shown in Table 3.

[0141] Table 3. Primer pairs for Taqman MGB probes for methylated DNA immunoprecipitation enrichment

[0142]

[0143]

[0144] 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.

[0145] 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.

[0146] The sequences of the Taqman MGB probe primer pairs after bisulfite conversion are shown in Table 4.

[0147] Table 4. Taqman MGB probe primer pairs after bisulfite conversion

[0148]

[0149] 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.

[0150] 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.

[0151] (4) Analysis of clinical sample test results

[0152] The offline data were analyzed, and 124 samples were enriched by methylated DNA immunoprecipitation method and bisulfite treatment DNA method, and then detected by qPCR.

[0153] Figure 2A-2D The following are examples of qPCR amplification curves of the same gastric cancer patient (sample number: GC106673) using the methylated DNA immunoprecipitation enrichment method and the bisulfite treatment DNA method. Figure 2A and 2B Respectively represent the qPCR amplification curves of OLIG2 and FERD3L genes after methylated DNA immunoprecipitation enrichment, with Ct values ​​of 33.96 and 34.32, respectively. Figure 2C and 2D The qPCR amplification curves of bisulfite-treated DNA for the two genes were 36.65 and 35.29, respectively. qPCR detection after immunoprecipitation enrichment of methylated DNA had obvious advantages.

[0154] 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 0.987 and 0.934, 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 = 650; 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 = 650. If the logistic scores of the OLIG2 and FERD3L 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 124 samples were statistically analyzed. The formula is as follows:

[0155] Formula I: logistic scores=e k / (1+e k )

[0156] Methylated DNA immunoprecipitation (Formula II): k = -2.91 × Ct OLIG2 -1.402×Ct FERD3L+152.908 Bisulfite conversion (Formula III): k = -1.09 × Ct OLIG2 -0.429×Ct FERD3L +56.945

[0157] Table 5 shows a comparison of qPCR detection based on the methylated DNA immunoprecipitation enrichment method and the gastroscopy results (gold standard), Table 6 shows a comparison of qPCR detection results based on bisulfite conversion treatment and the gastroscopy results, and Table 7 shows a comparison of the detection results based on qPCR detection based on the methylated DNA immunoprecipitation enrichment method and the bisulfite conversion treatment qPCR detection.

[0158] Table 5. Comparison of qPCR detection results based on methylated DNA immunoprecipitation enrichment method and gastroscopy results

[0159]

[0160] Table 6. Comparison of qPCR results based on bisulfite conversion treatment with gastroscopy results

[0161]

[0162] Table 7. Comparison of enrichment methods based on methylated DNA immunoprecipitation and bisulfite conversion

[0163]

[0164]

[0165] As shown in Tables 5 to 7, differentially methylated regions of the OLIG2 and FERD3L genes demonstrated a higher sensitivity (90.2%) for gastric cancer and a high specificity (96.8%) for non-gastric cancer samples when validated using a qPCR detection platform based on methylated DNA immunoprecipitation enrichment. The accuracy reached 93.5%, demonstrating overall superior performance to bisulfite conversion qPCR.

[0166] (5) Analysis of clinical gastric cancer staging sample test results

[0167] Among the 61 clinically diagnosed gastric cancer plasma samples, 16 samples were from gastric cancer stages 0-1, 11 samples were from gastric cancer stage II, 18 samples were from gastric cancer stage III, and 16 samples were from gastric cancer stage IV. The detection and statistical analysis of samples with different gastric cancer pathological stages using qPCR detection based on methylated DNA immunoprecipitation enrichment compared to qPCR detection based on bisulfite conversion treatment are shown in Tables 8 and 9:

[0168] Table 8. Detection of samples of different gastric cancer pathological stages

[0169]

[0170] Table 9. Statistical analysis of sensitivity of different gastric cancer pathological stages

[0171]

[0172] As can be seen from Tables 8 and 9, the differentially methylated regions of the OLIG2 and FERD3L genes also maintained a high sensitivity (87.5%, 90.9%) for gastric cancer stage 0-I and stage II samples when verified based on the methylated DNA immunoprecipitation enrichment method qPCR detection platform. The performance for early detection of gastric cancer is better than the bisulfite conversion treatment qPCR detection, providing a new potential marker for early detection of gastric cancer. Since the present invention maintains a high sensitivity in the early detection of gastric cancer, it can be used for the prediction of gastric cancer. 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 gastric cancer, 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.

[0173] 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 gastric cancer, characterized in that: It consists of the following two methylation molecular markers located in the human OLIG2 and FERD3L genes, respectively: chr21:33025814-33026014 and chr7:19145345-19145545, and was specifically mapped using hg38.

2. Use of a combination of methylation markers for gastric cancer in preparing a product, characterized in that: The product is used to diagnose or predict gastric cancer; the methylation marker combination for gastric cancer is composed of the following two methylation molecular markers located in the human OLIG2 and FERD3L genes, respectively: chr21:33025814-33026014 and chr7:19145345-19145545, which are specifically located through hg38.

3. The use according to claim 2, characterized in that The following steps are involved: A1, cfDNA samples were enriched by methylated DNA immunoprecipitation; A2, using the enriched product of step A1 as a template, perform fluorescence quantitative PCR amplification under primer and probe combination a to obtain Ct OLIG2 and Ct FERD3L 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 chr21:33025814-33026014 to obtain Ct OLIG2 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 chr7:19145345-19145545 to obtain Ct FERD3L ; A3. Process and judge the test results according to Formula I and Formula II; Formula I: logistic scores = e k / (1+e k ) Formula II: k = -2.91 × Ct OLIG2 -1.402×Ct FERD3L +152.908 A logistic score of ≤650 was considered negative for gastric cancer, and a logistic score of >650 was considered positive for gastric cancer; If the Ct value obtained by the fluorescent quantitative PCR test result is greater than 45 or no Ct value is detected, the Ct value is counted as 45 for substitution into Formula II.

4. The use according to claim 2, characterized in that The following steps are involved: B1, cfDNA samples were converted by bisulfite; B2, using the conversion product of step B1 as a template, and using primer and probe combination b, perform fluorescence quantitative PCR amplification to obtain Ct OLIG2 and Ct FERD3L Primer and probe combination b: primer pairs 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 chr21:33025814-33026014 to obtain Ct OLIG2 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 chr7:19145345-19145545 to obtain Ct FERD3L ; B3. Process and judge the test results according to Formula I and Formula III; Formula I: logistic scores = e k / (1+e k ) Formula III: k = -1.09 × Ct OLIG2 -0.429×Ct FERD3L +56.945 A logistic score of ≤650 was considered negative for gastric cancer, and a logistic score of >650 was considered positive for gastric cancer; If the Ct value obtained by the fluorescent quantitative PCR test result is greater than 45 or no Ct value is detected, the Ct value is counted as 45 for substitution into Formula III.

5. A kit for diagnosing or predicting gastric cancer, characterized in that: The kit is used to detect the methylation molecular marker combination according to claim 1; 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 in SEQ ID No. 1 and SEQ ID No. 2 and the Taqman MGB probe shown in SEQ ID No. 3, used for fluorescent quantitative PCR amplification of chr21: 33025814-33026014; the primer pair shown in SEQ ID No. 4 and SEQ ID No. 5 and the Taqman MGB probe shown in SEQ ID No. 6, used for fluorescent quantitative PCR amplification of chr7: 19145345-19145545; 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 chr21:33025814-33026014; 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 chr7:19145345-19145545.

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 gastric 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. OLIG2 and Ct FERD3L , use formula I and formula II to process and judge the test results; k=-2.91×Ct OLIG2 -1.402×Ct FERD3L +152.908 A logistic score of ≤650 was considered negative for gastric cancer, and a logistic score of >650 was considered positive for gastric 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 chr21:33025814-33026014 to obtain Ct OLIG2 The primer pair 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 chr7:19145345-19145545 to obtain Ct FERD3L .

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 gastric 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 OLIG2 and Ct FERD3L , 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 = -1.09 × Ct OLIG2 -0.429×Ct FERD3L +56.945 A logistic score of ≤650 was considered negative for gastric cancer, and a logistic score of >650 was considered positive for gastric 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 chr21:33025814-33026014 to obtain Ct OLIG2 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 chr7:19145345-19145545 to obtain Ct FERD3L .

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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