Reagent for esophageal cancer gene methylation detection and kit and application thereof

Abstract

The application belongs to the technical field of biology, and discloses a reagent for esophageal cancer gene methylation detection, a kit and application thereof, and specifically discloses a detection region for esophageal cancer gene methylation detection. The detection region for esophageal cancer gene methylation detection provided by the application can be used as an important detection index for early screening, process monitoring and prognosis evaluation of esophageal cancer, and DNA methylation abnormality is taken as a detection object. DNA methylation abnormality usually occurs in the early stage of cancer and runs through the occurrence and development process of cancer. Once the methylation state is formed, it needs to be continuously stimulated by the external environment for a long time to change. Therefore, the detection of the DNA methylation region can be used as an important biological index for early screening, process monitoring and prognosis evaluation of esophageal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to reagents, kits, and applications for the detection of gene methylation in esophageal cancer. Background Technology

[0002] Esophageal cancer refers to cancer originating from the esophageal epithelium, extending from the beginning of the hypopharynx to the esophagogastric junction. It is mainly classified into squamous cell carcinoma, adenocarcinoma, and undifferentiated carcinoma (less common, but highly malignant), and is one of the ten most common malignant tumors. The surgical resection rate for early-stage esophageal cancer is 100%, with a surgical mortality rate below 2.5%, and a 5-year survival rate of 92.6%. Early symptoms of esophageal cancer are often subtle. If progressive dysphagia, a feeling of obstruction, burning, stagnation, or fullness occurs after eating, it generally indicates an intermediate or advanced stage, and the 5-year survival rate for patients with intermediate or advanced esophageal cancer is only around 30%. Currently, auxiliary diagnostic methods for esophageal cancer mainly include imaging examinations (CT, upper gastrointestinal contrast radiography, MRI, PET-CT, ultrasound, etc.), endoscopy, and tumor marker testing. However, these techniques are either costly, invasive, or have low sensitivity, making them unsuitable for early screening of esophageal cancer patients. Therefore, there is an urgent need to find biomarkers for early esophageal cancer, develop sensitive, specific, and rapid detection technologies and methods, improve the early detection rate of esophageal cancer, enable more esophageal cancer patients to be diagnosed at an early stage, receive timely intervention and treatment, and reduce the mortality rate of esophageal cancer patients.

[0003] DNA methylation regulates cell proliferation, apoptosis, and differentiation, and is one of the earliest discovered and most thoroughly studied epigenetic regulatory mechanisms. Whether there is a necessary link between abnormal DNA methylation and tumorigenesis has been a hot topic in medical research. Currently, the only known form of DNA methylation in mammals is the methylation of the 5th carbon atom of cytosine in CpG dinucleotides. Abnormal gene methylation has been found in most human tumor tissues, and disorders in epigenetic coding in cancer cells are first manifested as disturbances in DNA methylation levels. Most abnormal methylation is hypermethylation of tumor suppressor genes; localized hypermethylation of CpG islands in tumor suppressor genes often leads to transcriptional silencing of these genes, which occurs earlier than the malignant proliferation of cancer cells and persists throughout the entire cancer development process. Esophageal cancer is a complex process involving the accumulation of multiple gene mutations, including abnormal methylation of various oncogenes and tumor suppressor genes. Therefore, the detection of DNA methylation indicators can be used for early tumor screening, diagnosis, grading, and monitoring the efficacy of anticancer drug treatment stages and prognosis.

[0004] Currently, methods for DNA methylation in tumor detection are mainly divided into two categories: whole-genome methylation analysis and site-specific methylation detection. Whole-genome methylation analysis, due to its high cost, is often used as a high-throughput screening method to identify target genes. Site-specific methylation detection methods can be further subdivided into restriction endonuclease assays combined with sodium bisulfite (COBRA), methylation-specific PCR (MSP), methylation-sensitive high-resolution melting curve analysis, and methylation quantitative fluorescence assay (MethyLight). Restriction endonuclease assays can only detect methylation at specific restriction sites, limiting their applicability. Methylation-specific PCR, based on conventional PCR and electrophoresis, is cumbersome and prone to sample contamination. Methylation-sensitive high-resolution melting curve analysis requires a fluorescence quantitative PCR instrument with a high-resolution melting (HRM) module, placing high demands on equipment. Methylation quantitative fluorescence assay, due to its high throughput and sensitivity, and the elimination of post-PCR electrophoresis and hybridization operations, reduces sample contamination and operational errors, and is widely used in DNA methylation detection. However, when using quantitative real-time PCR (qPCR) with methylation fluorescence, it relies on a standard curve or reference gene to determine the amount of nucleic acid, which can easily lead to intra- and inter-batch errors. Furthermore, its sensitivity for detecting low-concentration nucleic acid samples is insufficient, potentially causing false negatives and delaying patient diagnosis. Compared to qPCR, digital PCR offers higher detection sensitivity and accuracy through absolute quantitative counting of nucleic acid molecules. Digital PCR dilutes the nucleic acid sample, follows the Poisson distribution, and efficiently and sensitively completes the PCR amplification of the target nucleic acid fragment within a microreaction unit, acquiring the fluorescence signal and directly providing the copy number of the target sequence for statistical analysis. This achieves absolute quantification of the starting sample, reduces intra- and inter-batch errors in experimental results, improves detection sensitivity, and effectively reduces the occurrence of false negatives.

[0005] Based on the shortcomings of existing technologies, this invention establishes a digital PCR-based method for detecting esophageal cancer gene methylation by screening methylation genes related to esophageal cancer. The aim is to obtain detection reagents with higher sensitivity, specificity, and accuracy, enabling early screening, diagnosis, and classification of esophageal cancer patients, as well as monitoring the efficacy of anti-cancer drug treatment stages and prognosis. Summary of the Invention

[0006] The first aspect of the present invention is to provide a detection region for the detection of gene methylation in esophageal cancer.

[0007] A second aspect of the present invention aims to provide a combination of biomarkers for the detection of gene methylation in esophageal cancer.

[0008] A third aspect of the present invention is to provide a reagent.

[0009] A fourth aspect of the present invention is to provide a reagent kit.

[0010] The fifth aspect of this invention aims to provide a detection method for non-disease diagnostic purposes for detecting gene methylation in esophageal cancer.

[0011] The sixth aspect of this invention aims to provide the application of the first aspect of this invention for detecting esophageal cancer gene methylation regions, the second aspect of this invention for combining DNA methylation markers, the third aspect of this invention for reagents, or the fourth aspect of this invention for kits in products for the diagnosis and / or auxiliary diagnosis of esophageal cancer.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a gene methylation detection region for esophageal cancer, including the gene methylation detection regions shown in (a1) and / or (a2); (a1) The methylation detection region of the IRF4 gene, including the methylation detection region obtained by sulfite conversion of Chr6:391499-391683, the nucleotide sequence of Chr6:391499-391683 is shown in SEQ ID NO.22; (a2) The methylation detection region of the UNC5D gene, including the methylation detection region obtained by sulfite conversion of Chr8: 35235143-35235452, the nucleotide sequence of Chr8: 35235143-35235452 is shown in SEQ ID NO.23.

[0013] Preferably, the IRF4 gene methylation detection region described in (a1) includes Chr 6:391506-391630 and / or Chr 6: 391531-391683, wherein the nucleotide sequence of Chr 6:391506-391630 is shown in SEQ ID NO.24, and the nucleotide sequence of Chr 6: 391531-391683 is shown in SEQ ID NO.26.

[0014] Preferably, the UNC5D gene methylation detection region described in (b1) includes Chr8: 35235311-35235452 and / or Chr8: 35235310-35235426, wherein the nucleotide sequence of Chr8: 35235311-35235452 is shown in SEQ ID NO.27, and the nucleotide sequence of Chr8: 35235310-35235426 is shown in SEQ ID NO.28.

[0015] A second aspect of the present invention provides a DNA methylation biomarker combination for esophageal cancer detection, the biomarker combination comprising CpG island regions in the sequences of SEQ ID NO.22 and / or SEQ ID NO.23.

[0016] A third aspect of the present invention provides a reagent comprising a detection reagent capable of specifically detecting the methylation level of CpG dinucleotide sites in at least one of the target nucleotide sequences (b1)-(b3) in a biological sample; (b1) The full length of the nucleotide sequence shown in SEQ ID NO.22 and / or SEQ ID NO.23, or any portion thereof; (b2) The full length or any part thereof of the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.22 and / or SEQ ID NO.23; (b3) is at least 90% identical to (b1) or (b2) of the nucleotide sequence.

[0017] The inventors discovered that human esophageal cancer is associated with the DNA methylation level of the above-mentioned nucleotide sequence, and the methylation level in esophageal cancer samples is significantly higher than that in normal samples. By detecting the DNA methylation level of the above-mentioned nucleotide sequence, it is possible to provide a reference for whether the subject (object) has the risk of esophageal cancer, whether there is an early lesion of esophageal cancer, or whether esophageal cancer lesions have already occurred, thus providing a reference for the diagnosis or auxiliary diagnosis of esophageal cancer.

[0018] DNA methylation is the covalent bonding of a methyl group to the 5th carbon position of a cytosine CpG dinucleotide in the genome. DNA methylation level refers to the proportion of methylated CpG dinucleotide sites among all CpG dinucleotide sites in a specific nucleotide sequence or a portion thereof. In this invention, DNA methylation detection refers to determining whether each CpG island is methylated and calculating the methylation level in the nucleotide sequence of a specific region. In practical applications, different detection indicators can be used to compare DNA methylation levels depending on the specific circumstances. For example, in some cases, the Ct values ​​of the samples can be compared; in others, the methylation ratio of the marker in the sample can be calculated as (number of methylated molecules / (number of methylated molecules + number of unmethylated molecules)) × 100%, and then compared; in still others, statistical analysis and integration of various indicators are required to derive the final judgment index.

[0019] Preferably, the complementary sequence is a nucleotide sequence formed by a one-to-one correspondence between each base of the nucleotide sequence shown in SEQ ID NO.22 or SEQ ID NO.23.

[0020] Preferably, the partial region described in (b1) is the nucleotide sequence shown at positions 8-132 of SEQ ID NO.22 (i.e., the nucleotide sequence shown in SEQ ID NO.24 for Chr6:391506-391630 of the IRF4 gene), the nucleotide sequence shown at positions 32-185 of SEQ ID NO.22 (i.e., the nucleotide sequence shown in SEQ ID NO.26 for Chr6:391531-391683 of the IRF4 gene), the nucleotide sequence shown at positions 192-310 of SEQ ID NO.23 (i.e., the nucleotide sequence shown in SEQ ID NO.27 for Chr8:35235334-35235452 of the UNC5D gene), and / or the nucleotide sequence shown at positions 168-284 of SEQ ID NO.23 (i.e., the nucleotide sequence shown in SEQ ID NO.28 for Chr8:35235310-35235426 of the UNC5D gene).

[0021] Preferably, the nucleotide sequence described in (b3) is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to (b1) or (b2).

[0022] Preferably, the sequence with identity remains unchanged, maintaining the CpG dinucleotide site in the nucleotide sequence shown in SEQ ID NO.22 or SEQ ID NO.23, or its complementary sequence.

[0023] Preferably, the reagent further includes nucleic acid molecules.

[0024] Preferably, the nucleic acid molecule includes primer pairs capable of amplifying the nucleotide sequences shown in (b1), (b2) and / or (b3).

[0025] Preferably, the primer pair includes one or more of (c1)-(c4); (c1) The nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2; (c2) The nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8; (c3) The nucleotide sequences shown in SEQ ID NO.10 and SEQ ID NO.11; (c4) The nucleotide sequences shown in SEQ ID NO.13 and SEQ ID NO.14.

[0026] Preferably, the nucleic acid molecule further includes a probe capable of labeling the nucleotide sequences shown in (b1), (b2) and / or (b3).

[0027] Preferably, the probe comprises one or more of (d1)-(d4); (d1) The nucleotide sequence shown in SEQ ID NO.3; (d2) The nucleotide sequence shown in SEQ ID NO.9; (d3) The nucleotide sequence shown in SEQ ID NO.12; (d4) The nucleotide sequence shown in SEQ ID NO.15.

[0028] Preferably, the probe is labeled with modifying groups at both ends of its sequence, including 5' and 3' groups.

[0029] Preferably, the 5' group includes any one of FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3, and CY5.

[0030] Preferably, the 3' group includes any one of MGB, BHQ-1, BHQ-2, BHQ-3, and MGB-NFQ; more preferably, it is MGB.

[0031] Preferably, the biological sample includes at least one of a blood sample, a saliva sample, a tissue sample, and a cell sample derived from the esophagus.

[0032] Preferably, the tissue sample includes fresh pathological tissue or paraffin-embedded tissue, and the cell sample includes esophageal exfoliated cells.

[0033] Preferably, the biological sample includes an ex vivo biological sample derived from a mammal; more specifically, it may be derived from a human.

[0034] A fourth aspect of the present invention provides a reagent kit comprising the reagent of the third aspect of the present invention.

[0035] Preferably, the kit further includes reaction reagents capable of differentially modifying methylated and unmethylated DNA.

[0036] Preferably, the reaction reagent is a bisulfite.

[0037] Preferably, the kit further includes primer pairs and probes for the internal reference gene, quality control materials, and buffer solutions.

[0038] Preferably, the quality control products include positive quality control products and negative quality control products.

[0039] Preferably, the positive control includes esophageal cancer cell line DNA, and the negative control includes peripheral blood leukocyte DNA.

[0040] Preferably, the internal reference gene is GAPDH.

[0041] Preferably, the primer pair and probe of the internal reference gene include the primer pair described in SEQ ID NO.19 and SEQ ID NO.20, and the probe shown in SEQ ID NO.21.

[0042] Preferably, the esophageal cancer includes esophageal cancer at different stages, such as high-grade esophageal cancer, early-stage esophageal cancer, and advanced-stage esophageal cancer.

[0043] The different stages of esophageal cancer referred to in this invention can be based on the staging system defined by the American Joint Committee on Cancer (AJCC), such as esophageal squamous cell carcinoma. Stage 0 esophageal squamous cell carcinoma is TisN0M0, which presents as severe dysplasia or high-grade neoplasia of the esophagus. That is, even if a patient has stage 0 esophageal squamous cell carcinoma or an earlier stage, the kit of this invention can still maintain high sensitivity, reaching over 70%, and can be used for early screening of esophageal cancer, which is of great significance for early intervention and improving patient prognosis.

[0044] A fifth aspect of the invention provides a detection method for non-disease diagnostic purposes for detecting esophageal cancer gene methylation, comprising steps using a kit from the fourth aspect of the invention.

[0045] Preferably, the detection method specifically includes the following steps: preparing nucleic acid of the sample to be tested, converting it with bisulfite using a reaction reagent to obtain converted DNA, i.e., Bis-DNA; and detecting the methylation status of Bis-DNA using the kit of the third aspect of the present invention.

[0046] Preferably, the final concentration composition of the reaction system of the kit includes: 0.1-1 μM primer and 0.1-1 μM probe; more preferably, it includes 0.1-0.5 μM primer and 0.1-0.5 μM probe.

[0047] Preferably, the reaction conditions for detecting the methylation status of Bis-DNA using the kit are as follows: 90-95℃ for 10-15 min; 90-94℃ for 25-30 s, 56-62℃ for 60-65 s, 30-50 cycles; 95-98℃ for 8-10 min; further, 95℃ for 10 min; 94℃ for 30 s, 56℃ for 60 s, 45 cycles; 98℃ for 10 min.

[0048] Preferably, the sample to be tested includes at least one of a blood sample, a saliva sample, a tissue sample, and a cell sample derived from the esophagus.

[0049] Preferably, the tissue sample includes fresh pathological tissue or paraffin-embedded tissue, and the cell sample includes esophageal exfoliated cells.

[0050] Preferably, the esophageal cancer includes esophageal squamous cell carcinoma and esophageal adenocarcinoma.

[0051] Preferably, the esophageal cancer includes esophageal cancer at different stages, such as high-grade esophageal cancer, early-stage esophageal cancer, and advanced-stage esophageal cancer.

[0052] Preferably, the methylation status of Bis-DNA can be detected using methods known in the art, including but not limited to methylation-specific PCR, quantitative methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole-genome methylation sequencing, pyrosequencing, methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve analysis, methylation-sensitive restriction endonuclease assay, and quantitative fluorescence assay. Those skilled in the art can determine and prepare the detection reagents based on the reagents and tools required for known methods.

[0053] A sixth aspect of the present invention provides the use of the detection region of the first aspect of the present invention, the biomarker combination of the second aspect of the present invention, the reagent of the third aspect of the present invention, or the kit of the fourth aspect of the present invention in the preparation of products for the diagnosis and / or auxiliary diagnosis of esophageal cancer.

[0054] Preferably, the esophageal cancer includes esophageal squamous cell carcinoma and esophageal adenocarcinoma.

[0055] Preferably, the esophageal cancer includes esophageal cancer at different stages, such as high-grade esophageal cancer, early-stage esophageal cancer, and advanced-stage esophageal cancer.

[0056] The beneficial effects of this invention are: The detection region for esophageal cancer gene methylation detection provided by this invention can be used as an important detection indicator for early screening, progression monitoring, and prognostic assessment of esophageal cancer. It uses abnormal DNA methylation as the detection target. Abnormal DNA methylation usually occurs in the early stage of cancer and continues throughout the occurrence and development of cancer. Once its methylation state is formed, it requires continuous stimulation from the external environment for a long period of time to change. Therefore, the detection of DNA methylation region can serve as an important biomarker for early screening, progression monitoring, and prognostic assessment of esophageal cancer.

[0057] The reagents for DNA methylation detection and the kit for esophageal cancer gene methylation detection provided in this application can be used for the diagnosis and auxiliary diagnosis of esophageal cancer patients by detecting the DNA methylation level of specific nucleotide sequences. It can be used for esophageal cancer patients at different stages, including high-grade lesions, early esophageal cancer, and advanced esophageal cancer. Therefore, the detection kit of this application can be used for early screening of esophageal cancer. Its sensitivity can reach 70%, and its accuracy can reach more than 92%, or even 100%, showing good sensitivity and specificity. It is of great significance for early treatment intervention and improving patient prognosis.

[0058] Non-invasive detection: The esophageal cancer gene methylation detection kit provided by this invention can detect a variety of samples and can achieve non-invasive detection by detecting the gene methylation status of exfoliated esophageal cells.

[0059] High accuracy: The esophageal cancer gene methylation detection kit provided by this invention is based on digital PCR technology, which can efficiently and sensitively complete the PCR amplification of target nucleic acid fragments in a microreaction unit, obtain fluorescence signals for statistical analysis, completely eliminate the dependence on standard curves and directly give the copy number of the target sequence, improve the stability of experimental results within and between batches, achieve absolute quantification of starting samples, and improve the sensitivity of nucleic acid detection methods, effectively reducing the occurrence of false negatives. Attached Figure Description

[0060] Figure 1 A typical detection result diagram of digital PCR detection of esophageal cancer gene methylation provided for the implementation of this invention. Detailed Implementation

[0061] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0062] This application provides a reagent, kit, and application for detecting gene methylation in esophageal cancer. Detailed descriptions follow. It should be noted that the order of description in the following embodiments is not intended to limit the preferred order of the embodiments. Furthermore, in the description of this application, the term "comprising" means "including but not limited to".

[0063] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available. Experimental methods without specific conditions are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0064] This invention employs a bisulfite modification method to convert esophageal cancer nucleic acid samples for testing into bisulfite, combined with digital PCR technology. Candidate genes with significantly different methylation in esophageal cancer are screened using the TCGA database. Specific primers and probes for detecting esophageal cancer gene methylation are designed to amplify the bisulfite-modified DNA samples. The methylation status of the target gene in the sample is determined based on the number of positive droplets amplified by PCR, providing auxiliary diagnostic information for esophageal cancer.

[0065] In an exemplary embodiment of the present invention, digital PCR is used to detect the methylation status of Bis-DNA. The digital PCR employs droplet digital PCR technology. This involves adding the digital PCR mixture to a droplet generator to generate 10,000-100,000 micro-reaction droplets before PCR amplification. After the PCR amplification reaction, the type of fluorescence signal determines whether the sample contains methylated target DNA molecules. To determine the number and content of methylated target DNA molecules, an internal reference gene is set. Based on the formula: methylation ratio = methylated copy number / internal reference gene copy number × 100%, experimental results show that: a target gene methylation ratio ≥ 3% indicates a positive result; a target gene methylation ratio < 3% indicates a negative result.

[0066] Example 1: Sample DNA Extraction and Bisulfite Conversion 1. Sample DNA extraction DNA was extracted from esophageal exfoliated cells (esophageal exfoliated cells were provided by Henan Cancer Hospital) using nucleic acid extraction or purification reagent (general type) (catalog number: GE100) manufactured by Anhui Dajian Medical Technology Co., Ltd. The specific steps are as follows: a) Take an appropriate amount of cell sample, add 500 μL of lysis buffer and 30 μL of proteinase K, mix thoroughly and lyse at 70°C for 40 min; b) After a brief centrifugation, add 200 μL of isopropanol, mix thoroughly, transfer to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. d) Add 600 μL of rinsing solution I to rinse, centrifuge at 12000 rpm for 30 s, and discard the waste liquid; e) Add 600 μL of rinsing solution II and rinse, centrifuge at 12000 rpm for 30 s, then discard the waste liquid; f) Add 600 μL of rinsing solution II again to rinse, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and centrifuge at 12000 rpm for 3 min. g) Open the lid and air dry in a fume hood for 2 minutes; h) Add 60µL of eluent to the adsorption column, let stand at room temperature (20℃-30℃, the same below) for 3 min, and centrifuge at 12000rpm for 2 min; i) Repeat step h), collect the DNA into a centrifuge tube, and store at -20°C for later use.

[0067] 2. Bisulfite Conversion The genomic DNA obtained above was subjected to bisulfite conversion using nucleic acid extraction or purification reagent (centrifuge column type) (catalog number: ME100) produced by Anhui Dajian Medical Technology Co., Ltd., with the following steps: a) Take 45µL of the DNA sample to be tested (45ng / µL) into a new 1.5mL centrifuge tube and add 5µL of conversion buffer. Incubate in a metal bath at 37℃ for 15min. b) After incubation, add 100µL of the pre-prepared conversion solution to each sample, mix well and centrifuge briefly, then incubate in a metal bath at 50°C in the dark for 12-16 hours. c) Incubate the sample on ice (0-4℃) for 10 min; d) Place the adsorption column in the collection tube and add 400µL of binding solution to the adsorption column; e) Add the sample from step c to the adsorption column (containing binding solution), tighten the cap, invert and mix several times, centrifuge at full speed (14000 rpm) for 30 seconds, and discard the waste liquid. f) Add 100µL of washing solution to the adsorption column, centrifuge at full speed for 30s, and discard the waste liquid; g) Add 200µL of desulfurization solution to the adsorption column, incubate at room temperature (20℃~30℃) for 20min, then centrifuge at full speed for 30s and discard the waste liquid; h) Add 200µL of washing solution to the adsorption column, centrifuge at full speed for 30s, repeat adding 200µL of washing solution, centrifuge at full speed for 30s, and discard the waste liquid and collection tube. i) Place the adsorption column into a 1.5 mL sterile centrifuge tube, add 30 µL of elution buffer to the middle of the adsorption membrane, elute the transformed DNA, centrifuge at full speed for 1 min, collect Bis-DNA, and store at -20 °C for later use.

[0068] Example 2: Screening of differentially methylated genes and specific primers and probes in esophageal cancer tissue 1. Screening for differentially methylated genes in esophageal cancer tissue Methylation microarray data and corresponding transcriptome sequencing data related to esophageal cancer were obtained from the TCGA database and analyzed to screen for significantly different methylation sites. The results showed that IRF4 and UNC5D were significantly different methylation sites, and IRF4 and UNC5D were identified as differentially methylated genes in esophageal cancer tissue.

[0069] 2. Design and screening of specific primers and probes for esophageal cancer methylation detection 1) Screening of specific primers and probes Based on the nucleic acid sequences of the IRF4 and UNC5D genes and their promoter regions, the inventors, through repeated design, refinement, and screening, determined the genomic location of the IRF4 gene methylation detection region to be Chr6:391499-391683 (GRCh38 / hg38), which is the methylation detection region obtained after sulfite conversion of the nucleic acid sequence shown in SEQ ID NO.22. The genomic location of the UNC5D gene methylation detection region is Chr8:35235143-35235452 (GRCh38 / hg38), which is the methylation detection region obtained after sulfite conversion of the nucleic acid sequence shown in SEQ ID NO.23. Based on this, digital PCR probes and primers for the methylation of these genes were designed, with specific sequences shown in Table 1. Specific primers and probes for the internal reference gene GAPDH were also developed, with specific sequences shown in Table 2. The primers and probes were synthesized at Beijing Ruiboxingke Biotechnology Co., Ltd.

[0070] Table 1. Sequence information of primers and probes for IRF4 and UNC5D.

[0071] Table 2 Primer and probe sequence information for the internal reference gene GAPDH

[0072] The probe sequence described above is labeled with modifying groups at both ends, including a 5' group and a 3' group. The 5' group is selected from any one of FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3, and CY5, and the 3' group is selected from any one of MGB, BHQ-1, BHQ-2, BHQ-3, and MGB-NFQ.

[0073] 2) PCR amplification Further screening of primer-probe combinations was performed using PCR amplification (taking Bio-Rad QX200 microdroplet digital PCR as an example), as follows: Reaction system: 10 µL of 2× digital PCR reaction premix (Bio-Rad, catalog number: 1863023), 0.1 µL each of 10 µM GAPDH primers and probes, 0.5 µL each of primers and 0.2 µL of probes in a 10 µM primer-probe combination, 6 µL of Bis-DNA (prepared in Example 1), and water added to a final volume of 20 µL.

[0074] Droplet preparation: Add 70 µL of droplet generating oil (Bio-Rad Laboratories, catalog number: 1863005) to each 20 µL reaction system and place the card on a droplet generator to generate droplets. This process is generally completed within 2 minutes. Transfer the generated droplets to a 96-well plate and seal it using a preheated PX1 heat sealer. After sealing, the PCR reaction should be performed within 30 minutes. The PCR reaction procedure is shown in Table 3.

[0075] Table 3 PCR reaction procedure

[0076] The annealing temperature is selected from 56-62℃ based on the TM value of each primer combination.

[0077] Droplet Reading and Signal Analysis: Place the completed PCR 96-well plate into the droplet reader, open the QuantaSoft™ software, and create the sample module information according to the sample volume and layout. After setting up, run the program. After data reading, the threshold is automatically adjusted to assign positive and negative droplets to each detection channel. The sample adjustment threshold, methylation data collection, and analysis are displayed in the QuantaSoft™ software interface.

[0078] DNA samples from exfoliated esophageal cells of healthy individuals (DNA extraction and bisulfite conversion were the same as in Example 1) were used as a control group to simultaneously perform the above-mentioned PCR amplification.

[0079] The methylation ratio is calculated using the following formula: Methylation ratio = methylated copy number / internal reference gene copy number × 100%.

[0080] Using DNA samples of esophageal exfoliated cells (provided by Henan Cancer Hospital) from patients diagnosed with esophageal cancer and healthy individuals as templates, the primer-probe combinations selected in step 1) were screened by PCR amplification. The results showed that the methylation rate of the IRF4 gene was <3% (0.59%, 0.55%, and 0.56%, respectively) when testing the same normal esophageal exfoliated cell sample with primer-probe combination 1, 2, and 3, and the test results were all negative. However, when testing the exfoliated cells of the same esophageal cancer patient, the methylated copy number detected by primer-probe combination 2 (1324) was significantly less than that of primer-probe combination 1 (2298) and primer-probe combination 3 (2021), and the methylation rate of the IRF4 gene detected by primer-probe combination 2 was 18.85%, which was significantly lower than that of primer-probe combination 1 (30.70%) and primer-probe combination 3 (25.62%) (Table 4). Therefore, for the target gene IRF4, primer-probe combination 1 and primer-probe combination 3 were finally selected for subsequent experiments.

[0081] As shown in Table 5, when primer probe combinations 4, 5, and 6 were used to detect the same normal human endometrial exfoliated cell sample, the methylation rate of the UNC5D gene was <3% (0.93%, 0.84%, and 0.85%, respectively), and the test results were all negative. However, when detecting the same endometrial cancer patient's exfoliated cells, primer probe combination 6 detected a significantly lower number of methylated copies (3341) than primer probe combination 4 (5282) and primer probe combination 5 (4989), and its detected UNC5D gene methylation rate was 25.70%, which was significantly lower than that of primer probe combination 4 (44.41%) and primer probe combination 5 (49.56%). Therefore, for the target gene UNC5D, primer probe combination 4 and primer probe combination 5 were ultimately selected for subsequent experiments.

[0082] Table 4. Detection results of primer-probe combinations for the target gene IRF4

[0083] Table 5. Detection results of primer-probe combinations for the target gene UNC5D.

[0084] Example 3 A kit for detecting gene methylation in esophageal cancer comprises the following components: primer-probe combinations from Example 2 (at least one of primer-probe combination 1, primer-probe combination 3, primer-probe combination 4, or primer-probe combination 5), primers and probes for the internal reference gene GAPDH (same as in Example 2), negative control (human peripheral blood leukocyte DNA), positive control (human esophageal cancer cell line DNA), 2× digital PCR reaction premix, and bisulfite.

[0085] Example 4 Clinical Sample Testing The kit described in Example 3 was used to test clinical samples (tissue samples from 142 esophageal cancer patients, including 42 cases of high-grade lesions, 46 cases of early-stage esophageal cancer, and 54 cases of advanced-stage esophageal cancer, and 40 tissue samples from healthy individuals, all provided by Henan Cancer Hospital and pathologically identified) to verify the effectiveness of the kit. The sample pretreatment was the same as in Example 1, involving DNA extraction and bisulfite conversion. The reaction procedure was the same as in Example 2, section 2) PCR amplification. If the sum of the methylated copy number and the internal reference gene copy number of the tested sample was ≥100, the sample test result was valid and could continue analysis; if it was less than 100, the sample test was invalid and needed to be repeated. The methylation ratio of the sample was calculated using the following formula: Methylation ratio = methylated copy number / internal reference gene copy number × 100%. Experimental research results showed that: a target gene methylation ratio ≥3% was interpreted as a positive result; a target gene methylation ratio <3% was interpreted as a negative result. Typical test results are as follows: Figure 1 As shown.

[0086] 1. Use a kit containing a single primer-probe combination for single nucleic acid detection. Samples from 142 esophageal cancer patients and 40 healthy individuals were tested using primer probe combinations 1, 3, 4, or 5, respectively, to examine the sensitivity and specificity of kits containing only a single primer probe combination in tissue samples.

[0087] The results are shown in Table 6. Primer probe sets 1, 3, 4, and 5 showed a detection sensitivity greater than 92% for advanced esophageal cancer, greater than 73% for early esophageal cancer, and greater than 71% for high-grade esophageal lesions, indicating that the kit containing a single primer probe combination has high detection sensitivity for esophageal cancer patient tissues. Simultaneously, testing on tissue samples from healthy individuals showed that the kit containing a single primer probe combination had a detection specificity of 95%-100%, demonstrating high detection specificity.

[0088] Table 6. Sensitivity and specificity of kits containing single primer-probe combinations in tissue samples.

[0089] 2. Use a kit containing two primer-probe combinations for single nucleic acid detection. The kits containing any two primer probe combinations (primer probe combination 1, primer probe combination 3, primer probe combination 4, and primer probe combination 5) were used to perform single nucleic acid detection on samples from 142 esophageal cancer patients and 40 healthy individuals. The results of the single nucleic acid detection of the two primer probe combinations were superimposed and statistically analyzed to determine the detection result. A positive result was defined as a positive result for either primer probe combination. The sensitivity and specificity of the kit containing two primer probe combinations in tissue samples were then investigated.

[0090] The results are shown in Table 7. The kit containing two primer probe combinations showed a detection sensitivity of 96%-100% for advanced esophageal cancer, 80%-83% for early esophageal cancer, and 73%-79% for high-grade lesion samples. Meanwhile, the specificity for normal human samples was 92%-98%. This indicates that when the single nucleic acid detection results of the kit containing two primer probe combinations are statistically analyzed, the detection sensitivity is higher than that of the kit containing a single primer probe combination. However, the detection specificity is lower, suggesting that while the single nucleic acid detection method using kits containing two primer probe combinations increases sensitivity, it may lead to a decrease in specificity.

[0091] Table 7 shows the detection sensitivity and specificity of the kit containing a two-primer-probe combination in tissue samples.

[0092] 3. Perform dual nucleic acid detection using a kit containing two primer-probe combinations. The kits containing any two primer probe combinations (primer probe combination 1, primer probe combination 3, primer probe combination 4, and primer probe combination 5) were used to perform dual nucleic acid detection on samples from 142 esophageal cancer patients and 40 healthy individuals. Dual nucleic acid detection with two primer probe combinations was performed in a single tube. A positive result was defined as a positive result for either primer probe combination. The detection sensitivity and specificity of the kits containing two primer probe combinations in tissue samples were then investigated (Table 8).

[0093] The results showed that the dual nucleic acid detection using a kit containing two primer probe combinations had a sensitivity of 96%-99% for advanced esophageal cancer, 80%-83% for early esophageal cancer, and 73%-79% for high-grade lesions. The specificity for normal human samples was 95%-98%. This indicates that while the detection sensitivity of the dual nucleic acid detection kit containing two primer probe combinations was slightly lower than that of the single nucleic acid detection kit containing two primer probe combinations, its specificity was improved to some extent. Similarly, compared to the single nucleic acid detection kit containing a single primer probe combination, the detection specificity of the dual nucleic acid detection kit containing two primer probe combinations was slightly lower, but its detection sensitivity was significantly improved. Furthermore, the dual nucleic acid detection kit containing two primer probe combinations can significantly reduce reagent consumption, lower consumable costs, and reduce the workload for laboratory personnel, thus reducing labor costs.

[0094] Table 8 shows the results of dual nucleic acid detection using a kit containing a two-primer-probe combination.

[0095] In summary, the kit for esophageal cancer gene methylation detection provided in Example 3 has high detection sensitivity and specificity, making it an ideal choice for esophageal cancer diagnosis and early screening, thus contributing to the early diagnosis and treatment of esophageal cancer and showing good clinical application prospects.

[0096] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A marker combination for detection of esophageal cancer gene methylation, characterized by, The marker combination consists of CpG island regions from the sequences SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.

28.

2. An agent, characterized in that, This includes detection reagents capable of specifically detecting the methylation level of CpG dinucleotide sites in biological samples at SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.27, and SEQ ID NO.28; The detection reagent includes primer pairs and probes: The primer pairs include (c1) to (c4); (c1) The nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2; (c2) The nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8; (c3) The nucleotide sequences shown in SEQ ID NO.10 and SEQ ID NO.11; (c4) The nucleotide sequences shown in SEQ ID NO.13 and SEQ ID NO.14; The probes include (d1) to (d4): (d1) The nucleotide sequence shown in SEQ ID NO.3; (d2) The nucleotide sequence shown in SEQ ID NO.9; (d3) The nucleotide sequence shown in SEQ ID NO.12; (d4) The nucleotide sequence shown in SEQ ID NO.

15.

3. The agent of claim 2, wherein The probe has modifying groups labeled at both ends of its sequence.

4. A kit characterized in that, The kit comprises the reagent as described in claim 2 or 3.

5. The kit of claim 4, characterized in that The kit also includes reaction reagents that can differentially modify methylated and unmethylated DNA.

6. The kit of claim 5, wherein The kit also includes primer pairs and probes for the internal reference gene, quality control materials, and buffer solutions.

7. The kit of claim 6, wherein The primer pairs and probes for the internal reference gene include the primer pairs shown in SEQ ID NO.19 and SEQ ID NO.20, and the probe shown in SEQ ID NO.

21.

8. The use of the biomarker combination of claim 1, the reagent of any one of claims 2 to 3, or the kit of any one of claims 4 to 7 in the preparation of products for the diagnosis and / or auxiliary diagnosis of esophageal cancer.

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