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

Abstract

The application discloses a methylation molecular marker combination for gastric cancer diagnosis or prediction, which is composed of the following two methylation molecular markers respectively located at human OLIG2 and FERD3L genes: chr21:33025814-33026014 and chr7:19145345-19145545, and is specifically positioned by means of hg38. The application 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 OLIG2 and FERD3L gene methylation levels serve as markers for early diagnosis or prediction of gastric cancer, and enrich the selection of the skilled in the art.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology and gene detection, and particularly relates to a methylation marker combination for gastric cancer diagnosis or prediction, a kit and a computer readable storage medium. BACKGROUND

[0002] Gastroscopy, as the gold standard for the diagnosis of gastric cancer, has been widely used in China. However, due to its invasiveness, relatively high cost, and low acceptance among the population, it is difficult to be used for large-scale screening of gastric cancer in China. Moreover, gastroscopy is mainly performed on symptomatic patients, and its sensitivity and specificity are not ideal, resulting in a low rate of early cancer detection.

[0003] H. pylori infection is the most significant and common preventable cause of gastric cancer. On January 6, 2024, Wei Wenqiang et al. of the National Cancer Center, Peking Union Medical College, Chinese Academy of Medical Sciences, published a research paper on (Trends in the Burden of Risk Factors for Gastric Cancer in China, 2000-2050) in the Lancet. It is predicted that 40.7% of cardia gastric cancer and 62.1% of non-cardia gastric cancer cases will be caused by H. pylori infection by 2050. Although H. pylori infection is one of the main causes of gastric cancer, at least 90% of gastric cancer is associated with H. pylori infection, but not all infected individuals will develop gastric cancer. This means that H. pylori infection is a risk factor, but not the only determining factor.

[0004] Current common gastric cancer screening methods include serum pepsinogen (PG), serum gastrin, H. pylori (Hp) antibody detection, barium meal examination, and endoscopy. Existing screening guidelines recommend gastric cancer screening for high-risk populations aged ≥40 or 45 years. In China, there are more than 300 million people at high risk of gastric cancer, and for economic and medical resource considerations, it is urgent to stratify the screening population by risk before gastroscopy to identify high-risk populations and improve screening efficiency.

[0005] Peripheral blood is one of the most studied biological sample types. For asymptomatic, medium-risk, unwilling to undergo fecal examination or endoscopy, blood testing may be a popular choice. The current marketed products based on blood DNA detection for the auxiliary diagnosis of gastric cancer generally have a sensitivity of less than 85%, which cannot meet the clinical needs. Clinical practice shows that blood methylation detection is mainly limited by its relatively low sensitivity for identifying gastric cancer, which is 61.76% to 80.77%. Therefore, it is an urgent need to discover gastric cancer-related gene methylation markers in human blood samples based on non-bisulfite treatment and effectively detect the methylation level changes for early cancer screening.

[0006] Almost all tumors are caused and promoted by genetic alterations and epigenetic variations. By comparing tumor cells and normal cells, a large number of epigenetic abnormalities have been reported, of which DNA methylation is the most common epigenetic effect.

[0007] Methylation of cytosine on DNA is a covalent DNA "acquired" modification. DNA methylation is carried out by DNA methyltransferases (DNMTs). DNMTs can transfer a methyl group from a S-adenosyl methionine to the C-5 position of cytosine. DNA methylation almost exclusively occurs at CpG dinucleotide positions, which are unevenly distributed in the human genome, and the regions that are generally referred to as CpG islands. Such CpGs exist in repetitive sequences and regulatory regions of many genes 5' in the human genome. DNA methylation abnormalities that occur in tumors include hypomethylation (or demethylation) and hypermethylation, and hypermethylation occurs in, but is not limited to, tumor suppressor genes, and hypomethylation occurs in, but is not limited to, proto-oncogenes.

[0008] All aspects of tumor development and progression are likely to be associated with changes in DNA methylation, involving cell cycle regulation, DNA damage repair, biochemical metabolism of carcinogenic compounds, apoptosis, and angiogenesis. Different types of tumors can have a specific group of hypermethylated tumor suppressor genes and hypomethylated proto-oncogenes, i.e., a specific methylation profile for a cancer type, and the type of cancer can be identified according to the methylation profile.

[0009] Methylation enrichment technology is an analytical method for studying methylation modifications on DNA. DNA methylation is an important epigenetic modification involving the addition of a methyl group to the cytosine ring in DNA molecules. This modification plays a key role in biological processes such as regulation of gene expression, cell differentiation, and genome stability. Therefore, understanding the status of DNA methylation is very important 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-CpG Binding Domain sequencing), etc., in which:

[0011] MSP uses methylation-specific primers to selectively amplify methylated DNA fragments by PCR. It is simple and fast, and suitable for analysis of specific CpG sites, but cannot provide whole-genome methylation information, and is only suitable for previously determined target regions.

[0012] Methylation-sensitive restriction enzyme digestion uses the difference in sensitivity of restriction enzymes to DNA sequences to distinguish between methylated and non-methylated DNA regions. This method is based on the principle that DNA methylation affects the sensitivity of base pairs on the cytosine ring to restriction enzymes. This technique does not require the use of expensive sequencing technology and can be analyzed by methods such as gel electrophoresis. However, it cannot provide high-resolution information for individual CpG sites, and usually provides the methylation status of the overall region. At the same time, it is limited by the specificity of the selected restriction enzyme, and some methylation sites may be missed or overestimated. In addition, it cannot directly distinguish between 5-methylcytosine and other forms of DNA modification.

[0013] MeDIP-Seq uses methylation DNA antibodies to selectively enrich methylated DNA fragments, and then analyzes the enrichment by high-throughput sequencing technology. It can enrich the entire methylated genomic region and is suitable for whole-genome methylation analysis. However, it cannot provide high-resolution information for individual CpG sites.

[0014] MBD-Seq uses methylation DNA binding proteins (such as MBD2 or MBD3) to enrich methylated DNA fragments, and then analyzes by sequencing. It can provide higher enrichment efficiency and is suitable for whole-genome methylation analysis. However, similar to MeDIP-Seq, it cannot provide high-resolution information for individual CpG sites.

[0015] Methylation is also known as methylation conversion. Common methylation sequencing techniques include bisulfite sequencing (BS-seq). BS-seq uses bisulfite to treat DNA, converting unmethylated cytosine to uracil, while leaving methylated cytosine unaffected, and then analyzes the DNA through sequencing. It can provide high-resolution information on individual CpG sites and can perform methylation analysis across the entire genome. However, the experimental procedures are relatively complex.

[0016] Studies on early cancer screening have found that circulating cell-free DNA (cfDNA) methylation detection serves as a non-invasive, low-cost, sensitive, and accurate basis for early tumor detection, and can be used for cancer screening and various cancer classifications. Circulating tumor DNA (ctDNA) methylation is an important epigenetic modification. Gene methylation occurs before cancer develops and is a crucial mechanism in cancer development, acting as a "switch" regulating gene expression. Its stability and consistency are good, making it an ideal method for early cancer screening. Summary of the Invention

[0017] One of the technical problems to be solved by this invention is to provide a combination of methylation molecular markers for the diagnosis or prediction of gastric cancer, which are respectively located in human OLIG2 ( Oligodendrocyte Transcription Factor 2 ) and FERD3L ( Fer3 Like BHLH Transcription Factor The gene is composed of the following two methylation molecular markers: 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 combination of methylation markers for gastric cancer in the preparation of a product, the purpose of which is to diagnose or predict gastric cancer; the combination of methylation markers for gastric cancer consists 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 implementation schemes, the following steps are included:

[0020] A1 and cfDNA samples were enriched by methylated DNA immunoprecipitation.

[0021] A2. Using the enriched product from step A1 as a template, and under primer and probe combination a, perform real-time quantitative PCR amplification 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, for fluorescence quantitative PCR amplification of chr21:33025814-33026014 to obtain Ct OLIG2 ; 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, for fluorescence quantitative PCR amplification of chr21:33025814-33026014 to obtain Ct FERD3L ;

[0022] A3, processing and judging the detection results according to Formula I and Formula II;

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

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

[0025] logistic scores score ≤650 is determined as gastric cancer negative, and logistic scores score >650 is determined as gastric cancer positive;

[0026] The Ct value obtained from the fluorescence quantitative PCR detection result is >45 or no Ct value is detected, and the Ct value is all taken as 45 to be substituted into Formula II.

[0027] In some embodiments, the following steps are included:

[0028] B1, cfDNA sample is converted by bisulfite;

[0029] B2, taking the conversion product of step B1 as a template, performing fluorescence quantitative PCR amplification under primer and probe combination b to obtain Ct OLIG2 and Ct FERD3L ; 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, for fluorescence quantitative PCR amplification of chr21:33025814-33026014 to obtain Ct OLIG2; a primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11 and a Taqman MGB probe as shown in SEQ ID No. 12 for the fluorescence quantitative PCR amplification of chr7: 19145345-19145545 to obtain Ct FERD3L ;

[0030] B3, detecting result processing and judgment according to formula I and formula III;

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

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

[0033] The gastric cancer is determined as negative when the logistic scores score is less than or equal to 650, and the gastric cancer is determined as positive when the logistic scores score is greater than 650;

[0034] The Ct value obtained from the fluorescence quantitative PCR detection result is greater than 45 or the Ct value is not detected, and the Ct value is all taken as 45 to be substituted into formula III.

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

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

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

[0038] 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 for the fluorescent quantitative PCR amplification of chr21:33025814-33026014; primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12 for the fluorescent quantitative PCR amplification of chr7:19145345-19145545.

[0039] In some embodiments, the enrichment of methylated fragments is performed by using the Methylated DNA Immunoprecipitation (MeDIP) technology, and then the enriched DNA is used as a template for the fluorescent quantitative PCR under the condition of primer and probe combination a; the kit further comprises a methylated DNA antibody selected from one of 5-methylcytosine antibody, 5-mC antibody, 5-hmC antibody, 5-fC antibody, 5-caC antibody.

[0040] In some embodiments, one or more of the following are further included: 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 a MGB and a fluorescence quenching group, and the 5' end carries a fluorescence group; the combination formed by the fluorescence quenching group and the fluorescence group is selected from BHQ1 or NFQ and FAM, BHQ2 and VIC or HEX, BHQ2 and Cy3, BHQ2 and Cy5.

[0042] In some embodiments, the two Taqman MGB probes in primer and probe combination a respectively carry different fluorescence quenching group and fluorescence group combinations, so that in the fluorescent quantitative PCR amplification reaction with two Taqman MGB probes at the same time, the corresponding Ct value can be read according to the difference in fluorescence color.

[0043] In some embodiments, the two Taqman MGB probes in primer and probe combination b respectively carry different fluorescence quenching group and fluorescence group combinations, so that in the fluorescent quantitative PCR amplification reaction with two Taqman MGB probes at the same time, the corresponding Ct value can be read according to the difference in fluorescence color.

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

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

[0046] A fourth technical problem to be solved by the present application is to provide a computer readable storage medium comprising a program capable of being executed by a processor to realize analysis and processing of fluorescence quantitative PCR detection data of a methylation molecular marker combination as described above, to obtain a gastric cancer determination result, comprising the following steps:

[0047] Ct obtained by fluorescence quantitative PCR under the condition of primer and probe combination a, using DNA enriched by methylation DNA immunoprecipitation cfDNA as a template OLIG2 and Ct FERD3L , using formula I and formula II to process and judge the detection results;

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

[0049] logistic scores score ≤ 650 is determined as gastric cancer negative, and logistic scores score > 650 is determined as gastric cancer positive;

[0050] If the Ct value obtained by fluorescence quantitative PCR detection result is > 45 or no Ct value is detected, the Ct value is all taken as 45 to be substituted 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 ; primer pair as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6, used for fluorescence quantitative PCR amplification of chr7: 19145345-19145545 to obtain Ct FERD3L .

[0052] The application also provides another computer readable storage medium comprising a program capable of being executed by a processor to realize analysis and processing of fluorescence quantitative PCR detection data of the methylation molecular marker combination as described above to obtain a gastric cancer determination result, comprising the following steps:

[0053] Ct obtained by fluorescence quantitative PCR under the condition of primer and probe combination b using the bisulfite-converted DNA as a template OLIG2 and Ct FERD3L , and the detection result processing and judgment are performed by using formula I and formula III;

[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] logistic scores score ≤ 650 is determined as gastric cancer negative, and logistic scores score > 650 is determined as gastric cancer positive;

[0057] If the Ct value obtained by the fluorescence quantitative PCR detection result is > 45 or the Ct value is not detected, the Ct value is all taken as 45 to be substituted into formula III;

[0058] Primer and probe combination b: the primer pair as shown in SEQ ID No. 7 and SEQ ID No. 8 and the Taqman MGB probe as shown in SEQ ID No. 9 are used to obtain Ct OLIG2 by fluorescence quantitative PCR amplification of chr21: 33025814-33026014; the primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11 and the Taqman MGB probe as shown in SEQ ID No. 12 are used to obtain Ct FERD3L by fluorescence quantitative PCR amplification of chr7: 19145345-19145545.

[0059] In some embodiments, the program analysis and processing of the fluorescence quantitative PCR detection data of the computer readable storage medium come from the following fluorescence quantitative PCR reaction: the reaction system is 35 μL; the reaction liquid reagent used is 2 × Rapid TaqMaster Mix; and the reaction condition is 95℃ for 5 minutes, 95℃ for 15 seconds, 60℃ for 40 seconds, and amplification for 45 cycles.

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

[0061] The present application provides OLIG2, FERD3L gene methylation region (chr21:33025814-33026014, chr7:19145345-19145545, specific positioning by hg38) as a methylation molecular marker for gastric cancer diagnosis (including early diagnosis, auxiliary diagnosis) or prediction, enriching the selection of those skilled in the art.

[0062] The present application uses methylation DNA immunoprecipitation technology combined with fluorescence quantitative PCR to detect the methylation level of the methylation region of the OLIG2, FERD3L gene, which has high sensitivity, strong specificity, and very important clinical application value.

[0063] The present application is based on the methylation level of the specific region of the OLIG2, FERD3L gene, which can realize early detection of gastric cancer, and provides more biomarker (methylation molecular marker is one of the biomarkers) selection for gastric cancer screening and early diagnosis.

[0064] The concept, specific structure and technical effects of the present application will be further described below in combination with the drawings, so as to fully understand the purpose, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0065] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments or the prior art description will be briefly introduced below, and obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0066] Figure 1 The OLIG2, FERD3L methylation gene detection process in the embodiment 2 of the present application is shown.

[0067] Figures 2A-2D An amplification curve graph of a sample based on the methylation DNA immunoprecipitation enrichment method qPCR detection (as shown in Figure 2A and 2B ) and the amplification curve graph based on the bisulfite conversion treatment qPCR detection (as shown in Figure 2C and 2D ) are shown.

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

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

[0070] For the convenience of those skilled in the art, some terms appearing in this text are explained and described.

[0071] In this text, the singular forms "a", "an" and "the" include their plural forms unless the context clearly indicates otherwise. Thus, for example, "a reagent" can be understood to include a plurality of reagent components.

[0072] In this text, unless otherwise specified, the term "comprising", "including" or "containing" means containing the listed numerical values, steps or components, but also does not exclude the presence of other numerical values, steps or components.

[0073] In this text, "individual" 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] In this text, "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". Specifically, it refers to the area under the Receiver Operating Characteristic (ROC) curve. The ROC curve is a plot of the true positive rate against the false positive rate for the different possible cut-points of a diagnostic test. It shows the balance between sensitivity and specificity for a chosen cut-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 random test will have an ROC curve lying on the diagonal with an area of 0.5; reference: J. P. Egan (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0076] In order to detect the level changes of gastric cancer specific methylation genes in ctDNA, mine new gastric cancer methylation markers with higher sensitivity and specificity, construct an early screening and early diagnosis model, the inventors of the present application have made a lot of efforts to develop effective gastric cancer methylation markers in cfDNA, so that early diagnosis of cancer and cancer risk is possible. Unexpectedly, it was found that specific regions of OLIG2 and FERD3L genes were methylated in gastric cancer cells, and these specific regions of the genes were used as methylation markers to complete the present application.

[0077] In the present application, the inventors have unexpectedly found that the methylation level of OLIG2 and FERD3L genes is significantly different between gastric cancer and non-gastric cancer after long-term exploration and verification of a large number of clinical samples. OLIG2 is a major regulator of oligodendrocyte lineage specification, particularly regulating the critical stages 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, and the master regulator reprograms the epigenetic pattern genome-wide, thereby guiding the new transcriptome. 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 cancer is possible by analyzing the methylation of DNA related to a certain type of cancer. The current mainstream methylation analysis method is bisulfite treatment, which includes denaturation, deamination, and desulfonation. DNA is first denatured into single strands, then subjected to high temperature, high salt, acidic, and alkaline environments, and the resulting transformed DNA has the following characteristics: single-stranded, double-stranded mixed, fragment missing, gap damage, and uracil state 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 base transformation, there may be incomplete or excessive transformation of sequences, resulting in artificial bias, which is further amplified by subsequent PCR amplification, causing inaccurate signals. Therefore, the methylation markers obtained by bisulfite treatment generally have low sensitivity, especially in blood samples, where the number of free DNA fragments is already limited, and the difficulty of detecting methylation levels is greatly increased after bisulfite treatment.

[0079] An amplification curve is a graphical representation of the accumulation of PCR products during a polymerase chain reaction (PCR) process. It is generated by monitoring the increase in fluorescence signal in the reaction solution. The following are the characteristics of a typical PCR amplification curve:

[0080] Initial phase:

[0081] Threshold Cycle (Ct) value: In the early stages of the PCR reaction, the fluorescence signal may be low, but as the PCR products accumulate, the fluorescence signal gradually increases. The Ct value refers to the number of cycles required in the PCR reaction for the fluorescence signal to rise above a pre-set threshold value. The lower the Ct value, the higher the initial amount of target DNA in the sample.

[0082] Exponential growth phase:

[0083] Exponential phase: In the middle of the PCR reaction, the PCR product grows exponentially. At this time, the increase in Ct value will be accelerated, reflecting the exponential growth of the target DNA in the PCR reaction.

[0084] Plateau phase:

[0085] Plateau phase: In the later stage of the PCR reaction, the accumulation of PCR products reaches saturation and no longer grows exponentially. The PCR amplification curve of this stage forms a platform, and the increase in Ct value becomes slow.

[0086] In some embodiments of the present application, the diagnosis is early diagnosis, in particular, the early stage is stage 0-I or stage II of gastric cancer.

[0087] The solutions of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained commercially.

[0088] Example 1 Discovery of gastric cancer-specific methylation gene sites

[0089] In order to screen biomarkers that are specifically methylated in gastric cancer, this embodiment collects blood samples of 245 patients clinically diagnosed as gastric cancer (also known as gastric cancer positive samples), and blood samples of 306 patients clinically diagnosed as negative for gastric cancer.

[0090] 1. Preparation of methylation DNA sample library

[0091] (1) DNA extraction

[0092] The extraction of cfDNA was performed according to the instructions of the commercial extraction kit.

[0093] The concentration and fragment distribution of nucleic acids were controlled by Qubit 4.0 and Qsep 100, respectively. The yield of cfDNA extracted from 2 mL of human plasma should be greater than 5 ng, and cfDNA should have an enrichment peak at 167 bp or nearby. When the yield is greater than 50 ng, Qsep 100 capillary electrophoresis is used for fragment quality control, and when there is large fragment contamination, magnetic beads are used for fragment screening to remove large fragments.

[0094] (2) Library construction

[0095] The kit can be constructed with commercial library according to the method indicated in the instruction. The same type kits such as Rapid Plus DNA LibPrep Kit for illumina (Cat. No. RK20208, ABclonal) or VAHTS Universal Pro DNA Library Prep Kit for illumina Vazyme (Cat. No. ND608-02, Novozyme) are used for end repair, "A" tailing and connection with adapters. In this embodiment, VAHTS Universal Pro DNA Library Prep Kit for illumina Vazyme (Cat. No. ND608-02, Novozyme) is used for library construction.

[0096] (3) Methylation DNA immunoprecipitation

[0097] The methylation enrichment of cfDNA and tissue genomic DNA is carried out in different reactions, and each methylation enrichment reaction can complete 12-100 cfDNA library mixtures (about 10 ng of each cfDNA sample library is input) at the same time, and 10-24 genomic DNA library mixtures (about 100 ng of each genomic DNA library is input) can be completed at the same time. The methylation enrichment based on 5-methylcytosine (5mC) antibody principle uses commercial methylation enrichment kit or self-prepared reagent according to the instruction method. In this embodiment, zymo MeDIP kit (item number D5101-A) is used. Then purify the methylation enrichment reaction according to the instruction, and use the universal sequencing primer pair for 10-12 rounds of conventional PCR amplification to obtain a methylation DNA fragment library with a yield of more than 500 ng per reaction.

[0098] 2. Preparation of DNA probe library

[0099] (1) Probe design

[0100] The inventors selected the longest OLIG2 and FERD3L gene transcripts in the NCBI database to confirm the gene location. The sequence information in the promoter region, 5'UTR, the first exon (exon 1) and the upstream 1 kb of the start codon was screened and obtained.

[0101] The obtained OLIG2 and FERD3L gene target methylation regions were designed for probes. The design principles of the probes are as follows:

[0102] (1) Full coverage of target region without gap;

[0103] (2) No overlap;

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

[0105] The target region of the specific genome corresponding position and the probe coverage region of the specific genome corresponding position designed therefrom are shown in Table 1.

[0106] (2) Probe synthesis

[0107] According to the above probe design principle, 62 probes were designed for the methylation region of 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] 3. DNA capture probe hybridization

[0111] Liquid hybridization capture was performed using NadPrep hybridization capture reagent (Cat. No. REF1005101, Nanda). Hybridization capture reaction can be single or multiple. The total amount of MeDIP amplification library input for each hybridization capture reaction should be between 300 ng and 8 μg. After purification, 500 ng of library (less than 500 ng was all input), Human Cot DNA and NadPrep ® Nano Blockers were added, and the vacuum concentrator was preheated to 42°C. The rotation speed was set to 1000 rpm. After drying, the prepared hybridization reaction solution (containing the above probes panel) was added, and the system was shaken and centrifuged. The hybridization program was 95°C / 30 sec; 65°C / Hold (100°C hot cover), and the hybridization capture was performed for 4-16 hours. Then, the washed streptavidin magnetic beads were added to the hybridization system, and incubated for 40 minutes, with vortex mixing every 10 minutes to ensure complete resuspension of the magnetic beads. It is worth noting that the reaction temperature of hybridization capture is 65°C, which is different from the 63°C of the methylation probe designed based on bisulfite conversion.

[0112] After the completion of the hybridization capture reaction, the combined magnetic beads were washed with four kinds of washing solution provided in the kit. Each step required discarding the residual liquid. Finally, 20 μL of nuclease-free water was added, and the system was gently vortexed to mix.

[0113] 4. PCR amplification and purification after hybridization capture

[0114] PCR amplification of the product after hybrid capture, reagents using VAHTS Universal Pro DNA LibraryPrep Kit for illumina (Cat. No. ND608-02, Vazyme) in the amplification reagent for the experiment, the number of cycles in 12-13. After amplification, the same volume of VAHTS DNA Clean Beads (Cat. No. N411-03, Vazyme) is used to purify the product, and a relatively pure hybrid capture library is obtained; using Qubit4.0 for library concentration quantitative determination, and using Qsep100 full-automatic nucleic acid protein analyzer for library fragment size detection.

[0115] 5. Library sequencing and bioinformatics analysis

[0116] The library concentration needs to be diluted to 4nM, mixed according to the required data amount, and the total data amount should not exceed 120G. After mixing, 5μL of library was taken out, 5μL of 0.2N NaOH was added, and it was mixed by blowing, denatured for 5 minutes, and then 990μL of HT1 Buffer (REF:15058251, illumina) was immediately added after the end, vortexed, 105μL was taken out and added to 1295μL of HT1 Buffer, vortexed, and then the library for sequencing was obtained, with a concentration of 1.5pM.

[0117] The sequencer is NextSeq 550Dx produced by illumina, the reagent adopts High Output ReagentCartridge v2 (REF:15057929, illumina) (300 cycles), High Output Flow CellCartridge v2.5 (REF:20022408, illumina) and Buffer Cartridge v2 (REF:15057941, illumina), 1300μL of library for sequencing is added to the sample site of High Output ReagentCartridge v2, and each reagent is put in order, and the sequencing can start; this example adopts double-end sequencing, and the total length is about 30 hours.

[0118] 6. Quality control of sequencing data

[0119] The data was quality controlled using Fastp (version 0.22.0) to remove low-quality bases, and the overall Q20 of the clean data was more than 90%, and the Q30 was more than 85%. The average sequencing depth was about 300x. The average on-target rate of the probes in the above probe combination was more than 80%, which showed that the combination of methylation immunoprecipitation and liquid hybridization capture probes based on the present embodiment was feasible and effective for detecting cancer-related methylation regions.

[0120] 7. Analysis of gastric cancer-related OLIG2 and FERD3L gene methylation difference regions

[0121] The DiffBind tool (version 3.8.4) was used to screen the differential peaks of tumors and non-tumors, and two algorithms, DESeq and EdgeR, were used. The intersection was used for priority screening in the panel. The screening criteria were: 1) False Discovery Rate (FDR) <0.01, 2) Fold change <-1. After that, the most significant characteristic methylation regions of the gastric cancer group and the non-gastric cancer group that met the conditions were used as methylation difference regions.

[0122] In the present embodiment, from the OLIG2 and FERD3L gene regions covered by 62 probes, the most significant characteristic methylation regions of the gastric cancer group and the non-gastric cancer group were screened as methylation difference regions. The methylation CpG sites of these methylation difference regions are shown in Table 2.

[0123] Table 2, Methylation CpG sites of methylation difference regions

[0124]

[0125] The RPM index (Reads per million mapped reads, i.e., reads per million mapped reads) of the methylation difference regions (reads region covering the CpG sites in Table 2) in 245 gastric cancer positive samples and 306 gastric cancer negative samples was analyzed. The P value was less than 0.005, and the difference was significant.

[0126] Example 2: Detection of OLIG2 and FERD3L methylation genes in clinical samples and comparison of different treatment schemes

[0127] To further verify the clinical performance of the gastric cancer related OLIG2 and FERD3L gene methylation difference region on the plasma samples of gastric cancer, the inventors detected another two groups of samples (61 plasma samples clinically diagnosed as gastric cancer and 63 plasma control samples with negative gastroscopy) using qPCR method. Among the above 61 plasma samples clinically diagnosed as gastric cancer, there were 16 samples of gastric cancer 0-I stage, 11 samples of gastric cancer II stage, 18 samples of gastric cancer III stage, and 16 samples of gastric cancer IV stage.

[0128] The OLIG2 and FERD3L methylation gene detection flowchart is shown in Figure 1 Specifically:

[0129] (1) DNA extraction

[0130] The extraction of cfDNA was performed using a commercial kit according to the instructions.

[0131] Qubit4.0 and Qsep100 were used to control the concentration and fragment distribution of nucleic acids. The yield of cfDNA extracted from 4 mL of human plasma should be greater than 10 ng, and cfDNA should have an enrichment peak at 167 bp or nearby. When the yield is greater than 50 ng, Qsep100 capillary electrophoresis is required for fragment quality control, and when there is a large fragment contamination, magnetic beads are required for fragment screening to remove large fragments.

[0132] (2) Methylation DNA treatment

[0133] ① Methylation DNA immunoprecipitation

[0134] Take 1 / 2 of the total amount of extracted nucleic acids, perform cfDNA methylation enrichment, and divide into different reactions. The methylation enrichment based on the principle of 5mC antibody uses zymoMeDIP kit (item number D5101-A), and the methylation enrichment reaction is purified according to the instructions, with an elution volume of 50 μL.

[0135] ② Methylated DNA bisulfite conversion

[0136] Take 1 / 2 of the total amount of extracted nucleic acids, perform cfDNA methylation bisulfite treatment, and divide into different reactions. The methylation DNA treatment based on the principle of bisulfite conversion uses DNA conversion kit (EZ DNA Methylation Kit, D5002) of ZYMO RESEARCH Bio Company to perform DNA bisulfite treatment. The elution volume is 50 μL.

[0137] (3) qPCR detection

[0138] The primer probe was synthesized by Shanghai Bailingge Biotechnology Co., Ltd., and the specific sequence information is as follows:

[0139] The sequence of the methylation DNA immunoprecipitation enrichment Taqman MGB probe primer pair is shown in Table 3.

[0140] Table 3, methylation DNA immunoprecipitation enrichment Taqman MGB probe primer pair

[0141]

[0142] The 3' end of the probe is labeled with MGB, the 5' end is labeled with a fluorescent group (FAM), and the 3' end is labeled with a quenching group (BHQ1).

[0143] Using the methylation DNA immunoprecipitation enrichment as a template, PCR amplification was performed, and the final concentration of each primer was 10 μM. Each gene was amplified by single reaction. The PCR reaction system was 5 μL of enriched template DNA, 2.5 μL of premix containing the above primers; PCR reaction reagent (2×Rapid Taq Master Mix) 17.5 μL, and the total volume was supplemented with water to 35 μL. The PCR reaction conditions are as follows: 95℃ for 5 minutes, 95℃ for 15 seconds, 60℃ for 40 seconds, and 45 cycles of amplification.

[0144] The sequence of the Taqman MGB probe primer pair after bisulfite conversion is shown in Table 4.

[0145] Table 4, Taqman MGB probe primer pair after bisulfite conversion

[0146]

[0147] The 3' end of the probe is labeled with MGB, the 5' end is labeled with a fluorescent group (FAM), and the 3' end is labeled with a quenching group (BHQ1).

[0148] Using the DNA after bisulfite conversion as a template, PCR amplification was performed, and the final concentration of each primer was 10 μM. Each gene was amplified by single reaction. The PCR reaction system was 5 μL of enriched template DNA, 2.5 μL of premix containing the above primers; PCR reaction reagent (2×Rapid Taq Master Mix) 17.5 μL, and the total volume was supplemented with water to 35 μL. The PCR reaction conditions are as follows: 95℃ for 5 minutes, 95℃ for 15 seconds, 60℃ for 40 seconds, and 45 cycles of amplification.

[0149] (4) Analysis of clinical sample detection results

[0150] The data were analyzed, and 124 samples were detected by methylation DNA immunoprecipitation enrichment method and bisulfite DNA method, and then qPCR was used for detection.

[0151] Figures 2A-2D The sample of the same gastric cancer patient (sample number: GC106673) was detected by methylation DNA immunoprecipitation enrichment method and bisulfite DNA method, and the qPCR amplification curve was shown, wherein Figure 2A and 2B OLIG2 and FERD3L gene methylation DNA immunoprecipitation enrichment qPCR amplification curve, Ct value was 33.96, 34.32, respectively, Figure 2C and 2D Two genes bisulfite DNA qPCR amplification curve, Ct value was 36.65, 35.29, respectively, and the methylation DNA immunoprecipitation enrichment qPCR detection had obvious advantages.

[0152] The Ct value of the sample with Ct value> 45 or undetermined Ct value (Undetermined) was set to 45, and the logistic regression formula was used for calculation, and the ROC curve was drawn according to the calculation results. As shown in Figure 3A and Figure 3B The area under the curve AUC of the ROC curve based on two different methods was 0.987 and 0.934, respectively. According to the ROC curve, the threshold (cut-off value) was set for different methods: based on methylation DNA immunoprecipitation enrichment method qPCR detection (i.e. methylation DNA immunoprecipitation fluorescence quantitative PCR method), the cut-off value was set to logistic scores=650; based on bisulfite conversion qPCR detection (i.e. bisulfite conversion fluorescence quantitative PCR method), the cut-off value was set to logistic scores=650. If the amplification logistic scores value of the two genes OLIG2 and FERD3L of the sample to be detected is equal to or lower than the set cut-off value, the sample is determined to be negative, otherwise it is determined to be positive. Thus, the detection results of 124 samples were statistically analyzed. The formula is as follows:

[0153] Formula I: logistic scores=e k / (1+e k )

[0154] Methylation DNA immunoprecipitation (formula II): k=-2.91×Ct OLIG2 -1.402×Ct FERD3L +152.908

[0155] Bisulfite conversion (Formula III): k = -1.09 x Ct OLIG2 -0.429 x Ct FERD3L +56.945

[0156] Table 5 shows the comparison of the qPCR detection based on the methylation DNA immunoprecipitation enrichment method with the gastroscopy results (gold standard), Table 6 shows the comparison of the qPCR detection based on the bisulfite conversion treatment with the gastroscopy results, and Table 7 shows the comparison of the detection results of the qPCR detection based on the methylation DNA immunoprecipitation enrichment method and the qPCR detection based on the bisulfite conversion treatment.

[0157] Table 5, Comparison of qPCR detection results based on the methylation DNA immunoprecipitation enrichment method with gastroscopy results

[0158]

[0159] Table 6, Comparison of qPCR detection results based on the bisulfite conversion treatment with gastroscopy results

[0160]

[0161] Table 7, Comparison of the methylation DNA immunoprecipitation enrichment method with the bisulfite conversion treatment

[0162]

[0163] As can be seen from Tables 5 to 7, the OLIG2 and FERD3L gene methylation difference regions have higher sensitivity (90.2%) to gastric cancer and higher specificity (96.8%) to non-gastric cancer samples when verified by the qPCR detection platform based on the methylation DNA immunoprecipitation enrichment method, and the accuracy reaches 93.5%. The overall performance is better than that of the qPCR detection based on the bisulfite conversion treatment.

[0164] Analysis of detection results of clinical gastric cancer staging samples

[0165] Among the above-mentioned 61 plasma samples clinically diagnosed as gastric cancer, there are 16 samples of gastric cancer 0-I stage, 11 samples of gastric cancer II stage, 18 samples of gastric cancer III stage, and 16 samples of gastric cancer IV stage. The detection and statistical analysis of different gastric cancer pathological staging samples based on the qPCR detection based on the methylation DNA immunoprecipitation enrichment method and the qPCR detection based on the bisulfite conversion treatment are shown in Tables 8 to 9:

[0166] Table 8, Detection of different gastric cancer pathological staging samples

[0167]

[0168] Table 9, sensitivity statistics of different gastric cancer pathological stages

[0169]

[0170] As shown in Tables 8-9, the OLIG2 and FERD3L gene methylation difference regions maintain high sensitivity (87.5%, 90.9%) in the validation of the methylation DNA immunoprecipitation enrichment method qPCR detection platform for gastric cancer 0-I and II stage samples, and the performance for early gastric cancer detection is better than that of the bisulfite conversion treatment qPCR detection, which provides a new potential marker for early gastric cancer detection. Since the present application maintains high sensitivity in early gastric cancer detection, it can be used for the prediction of gastric cancer. The present application uses cfDNA as the analysis sample, which is easy to obtain, and since cfDNA contains a lot of information, it is not 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, and has the characteristics of being superior to single tissue samples in the context of high-throughput sequencing analysis.

[0171] The principles and implementation manners of the present application are described by applying specific examples in the present text, and the above examples are only used to help understand the method of the present application and its core idea; meanwhile, for those skilled in the art, according to the idea of the present application, changes will be made in the specific implementation manners and application ranges, and the changes and improvements of the present application will be possible without exceeding the concept and range defined by the claims, and the content of the examples in the present specification should not be understood as the limitation of the present application.

Claims

1. A methylation molecular marker combination for diagnosis or prediction of gastric cancer, characterized by, consist of the following 2 methylation molecular markers respectively located in human OLIG2 and FERD3L genes: chr21:33025814-33026014 and chr7:19145345-19145545, specifically located by hg38.

2. Use of a primer and probe combination of a methylation marker combination for detecting gastric cancer in the manufacture of a product, characterized in that, The use of the product is for diagnosing or predicting gastric cancer; the methylation marker combination of the gastric cancer consists of the following 2 methylation molecular markers respectively located in human OLIG2 and FERD3L genes: chr21:33025814-33026014 and chr7:19145345-19145545, specifically located by hg38.

3. Use according to claim 2, wherein the compound is ###0002### The method comprises the following steps: A1, the cfDNA sample is enriched by methylation DNA immunoprecipitation; A2. Perform a fluorescent quantitative PCR amplification with the enriched product of step Al as a template, 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 for fluorescent quantitative PCR amplification of chr21:33025814-33026014 to obtain Ct OLIG2 ; primer pair as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6 for fluorescent quantitative PCR amplification of chr7:19145345-19145545 to obtain Ct FERD3L ; A3, the detection result processing and judgment are performed according to formula I and formula II; Formula I: logistic scores = e k / (1 + e k ) Equation II: k = -2.91 x Ct OLIG2 -1.402 x Ct FERD3L +152.908 The gastric cancer is determined as negative when the logistic scores score is ≤650, and as positive when the logistic scores score is >650; The Ct value obtained from the fluorescence quantitative PCR detection result is >45 or the Ct value is not detected, and the Ct value is all taken as 45 to be substituted into formula II.

4. The use according to claim 2, wherein the compound is ###0002### The method comprises the following steps: B1, the cfDNA sample is subjected to bisulfite conversion; B2. Perform a fluorescent quantitative PCR amplification with the conversion product of step B1 as template, under primer and probe combination b to obtain Ct OLIG2 and Ct FERD3L ; primer and probe combination b: primer pair as depicted in SEQ ID No. 7 and SEQ ID No. 8 and Taqman MGB probe as depicted in SEQ ID No. 9 for fluorescent quantitative PCR amplification of chr21 :33025814-33026014 to obtain Ct OLIG2 ; primer pair as depicted in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as depicted in SEQ ID No. 12 for fluorescent quantitative PCR amplification of chr7:19145345-19145545 to obtain Ct FERD3L ; B3, the detection result processing and judgment are performed according to formula I and formula III; Formula I: logistic scores = e k / (1 + e k ) Equation III: k = -1.09 x Ct OLIG2 - 0.429 x Ct FERD3L + 56.945 The gastric cancer is determined as negative when the logistic scores score is ≤650, and as positive when the logistic scores score is >650; The Ct value obtained from the fluorescence quantitative PCR detection result is >45 or the Ct value is not detected, and the Ct value is all taken as 45 to be substituted into formula III.

5. A kit for diagnosis or prognosis of gastric cancer, characterized by, The kit is used for detecting the methylation molecular marker combination according to claim 1; The kit comprises primer and probe combination a and / or primer and probe combination b; Primer and probe combination a: a primer pair as shown in SEQ ID No. 1 and SEQ ID No. 2 and a Taqman MGB probe as shown in SEQ ID No. 3, used for fluorescence quantitative PCR amplification of chr21:33025814-33026014; a primer pair as shown in SEQ ID No. 4 and SEQ ID No. 5 and a Taqman MGB probe as shown in SEQ ID No. 6, used for fluorescence quantitative PCR amplification of chr7:19145345-19145545; 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 for the fluorescent quantitative PCR amplification of chr21:33025814-33026014; primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12 for the fluorescent quantitative PCR amplification of chr7:19145345-19145545.

6. The kit of claim 5, wherein Also included are one or more of the following: 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 of claim 5, wherein The 3' end of the Taqman MGB probe is provided with a MGB and a fluorescence quenching group, and the 5' end is provided with a fluorescence group; the combination formed by the fluorescence quenching group and the fluorescence 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 program can be executed by a processor to achieve analysis and processing of the fluorescent quantitative PCR detection data of the methylation molecular marker combination according to claim 1, to obtain a gastric cancer determination result, including the following steps: Ct obtained by performing the fluorescent quantitative PCR under the condition of primer and probe combination a using the DNA enriched by methylated DNA immunoprecipitation cfDNA as a template OLIG2 and Ct FERD3L , and the detection result is processed and judged by using formula I and formula II; Formula I: logistic scores = e k / (1 + e k ) Equation II: k = -2.91 x Ct OLIG2 -1.402 x Ct FERD3L +152.908 logistic scores score ≤ 650 is determined as gastric cancer negative, and logistic scores score > 650 is determined as gastric cancer positive; If the Ct value obtained from the fluorescent quantitative PCR detection result is > 45 or no Ct value is detected, the Ct value is all taken as 45 to be substituted 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 for the fluorescent quantitative PCR amplification of chr21 :33025814-33026014 to obtain Ct OLIG2 ; primer pair as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6 for the fluorescent quantitative PCR amplification of chr7:19145345-19145545 to obtain Ct FERD3L .

9. A computer-readable storage medium, characterized in that, The program can be executed by a processor to achieve analysis and processing of the fluorescent quantitative PCR detection data of the methylation molecular marker combination according to claim 1, to obtain a gastric cancer determination result, including the following steps: Ct obtained from the fluorescent quantitative PCR under the condition of primer and probe combination b using the DNA converted by the bisulfite as the template OLIG2 and Ct FERD3L , and the detection result is processed and judged by using formula I and formula III; Formula I: logistic scores = e k / (1 + e k ) Equation III: k = -1.09 x Ct OLIG2 - 0.429 x Ct FERD3L + 56.945 logistic scores score ≤ 650 is determined as gastric cancer negative, and logistic scores score > 650 is determined as gastric cancer positive; If the Ct value obtained from the fluorescent quantitative PCR detection result is > 45 or no Ct value is detected, the Ct value is all taken as 45 to be substituted 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 for the fluorescent quantitative PCR amplification of chr21 :33025814-33026014 to obtain Ct OLIG2 ; primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12 for the fluorescent quantitative PCR amplification of chr7:19145345-19145545 to obtain Ct FERD3L .

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

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