Methylated biomarkers for detecting esophageal cancer and uses thereof

Abstract

The invention discloses a methylated biomarker for detecting esophageal cancer and application of the methylated biomarker. The invention provides a methylated biomarker, and the methylated biomarker comprises any one selected from the following differential methylated regions or any combination of the differential methylated regions: chr17: 49021947-49022096, chr19: 11998652-11998772, and the like. The invention also provides a method for preparing the methylated biomarker, and the methylated biomarker comprises any one selected from the following differential methylated regions: chr17: 49021947-49022096, chr19: 11998652-11998772, and the like. The methylated biomarker provided by the invention can be used for diagnosing esophageal cancer, and has good sensitivity, specificity and accuracy.

Description

[0001] Priority and related applications

[0002] This application claims priority to Chinese patent application 202311621010.2, filed on November 29, 2023, entitled “Methylation biomarkers or combinations and applications thereof for detecting esophageal cancer”, and all contents of that application, including the appendix, are incorporated into this application by reference. Technical Field

[0003] The present invention belongs to the field of biotechnology, and in particular relates to a methylation biomarker for detecting esophageal cancer and a use thereof. Background Art

[0004] Esophageal cancer is one of the most common malignant tumors in the world. According to the global cancer statistics in 2020, the number of new cases of esophageal cancer reached 604,000 and the number of deaths reached 544,000. China is a high-incidence area of ​​esophageal cancer. Although the incidence and mortality of esophageal cancer in China are both on a downward trend, it is still the main malignant tumor threatening the health of Chinese residents. According to the prevalence of malignant tumors in China in 2015, there were 246,000 new cases of esophageal cancer in my country, the crude incidence of esophageal cancer in my country was 17.8 / 100,000, the crude incidence in urban areas was 12.6 / 100,000, and the crude incidence in rural areas was 24.6 / 100,000; there were 188,000 deaths from esophageal cancer, the crude mortality rate of esophageal cancer in my country was 13.7 / 100,000, the crude mortality rate in urban areas was 10.0 / 100,000, and the crude mortality rate in rural areas was 18.4 / 100,000. The incidence and mortality rates ranked sixth and fourth among all malignant tumors, respectively. Esophageal cancer is extremely invasive and has a low survival rate, with a 5-year survival rate of about 15%-25%. China, especially the northern region of China, has the highest incidence and mortality rates of esophageal cancer in the world, and the prognosis is very poor.

[0005] Currently, gastrointestinal endoscopy is still the main means of diagnosing esophageal cancer. However, since gastrointestinal endoscopy is an invasive examination, the operation is relatively complicated, which will cause certain pain to the patient and greatly affect the patient's physical and mental health. It is also accompanied by the risk of infection and bleeding.

[0006] In recent years, with the development of high-throughput sequencing technology, scholars at home and abroad have conducted increasingly in-depth research on cancer. Among them, the technology of non-invasive screening using methylation signals of free DNA (cfDNA) in body fluids is very important. Since DNA methylation occurs in the early stages of the tumor and is tissue-specific, it is one of the best choices for tracking early tumor signals.

[0007] Therefore, there is still a need to develop new DNA methylation biomarkers to identify early esophageal cancer, assist in accurate clinical diagnosis and guide treatment. Summary of the invention

[0008] One of the objectives of the present invention is to provide a methylation biomarker for detecting esophageal cancer. The methylation biomarker can be used for the detection of early esophageal cancer.

[0009] The technical solution to achieve the above purpose is as follows:

[0010] In a first aspect of the present invention, a methylation biomarker is provided, wherein the methylation biomarker comprises any one or any combination selected from the following differentially methylated regions:

[0011] <h2 style=";text-align:left;direction:ltr">chr17:49021947-49022096,chr19:11998652-11998772,chr7:37487534-37487813,chr7:64349109-64349229,chr19:11998775-11998895,chr6 :28303840-28303960、chr7:87229481-87229905、chr14:60385938-6038 6058、chr9:35757267-35757387、chr11:86383212-86383675、chr12:220 94867-22095025、chr3:99594764-99594983、chr14:60386224-60386387 、chr17:36103848-36104221、chr17:59539346-59539760、chr14:600971 25-60097295、chr19:56989290-56989474、chr1:200011526-200011646、 chr12:54383600-54383720、chr17:7348591-7348915、chr17:36103471-3 6103693,chr10:94831568-94832005,chr19:56915342-56915462,chr7:37488070-37488477,chr19:56904581-56904938,chr17:37321401-3732 1702, chr7:87229906-87230345, chr8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-79627190, chr5:158532400-158532520 、chr2:131792932-131793052、chr3:194208336-194208456、chr17:4667 3964-46674084、chr4:147560771-147561088、chr12:133464324-133464 463, chr19:58038983-58039194, chr3:157812609-157812815, chr7:37960353-37960473, chr6:21667550-21667670, chr10:97804121-97804338chr20:61560374-61560535, chr6:78173165-78173373, chr1:8277528-8277747, chr17:29298072-29298242, chr7:27196138-2719 6564, chr18:67068779-67069168, chr3:69591929-69592195, chr3:59035661-59035943, chr15:41805632-41805752, chr2:6328535 8-63285536, chr4:96471066-96471186, chr20:23029625-23030082, chr1:243646395-243646735, chr16:68544226-68544346, chr5 :35925138-35925258, chr11:44331133-44331538, chr10:20105505-20105717, chr1:18971873-18972006, chr6:1384523-1384807. ,

[0012] In some specific embodiments, the differentially methylated region includes at least one of chr17:49021947-49022096 and chr19:11998652-11998772.

[0013] Furthermore, the differentially methylated region also includes at least one of: chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895 and chr6:28303840-28303960.

[0014] Furthermore, the differentially methylated region also includes at least one of chr7:87229481-87229905 and chr14:60385938-60386058.

[0015] Furthermore, the differentially methylated region also includes at least one of chr9:35757267-35757387 and chr11:86383212-86383675.

[0016] Furthermore, the differentially methylated region also includes at least one of chr12:22094867-22095025 and chr3:99594764-99594983.

[0017] Furthermore, the differentially methylated region also includes at least one of: chr14:60386224-60386387, chr17:36103848-36104221, chr17:59539346-59539760, chr14:60097125-60097295 and chr19:56989290-56989474.

[0018] Furthermore, the differentially methylated region also includes: at least one of chr1:200011526-200011646, chr12:54383600-54383720, chr17:7348591-7348915 and chr17:36103471-36103693; optionally, the differentially methylated region also includes chr10:94831568-94832005.

[0019] Furthermore, the differentially methylated regions also include: at least one of chr9:79627070-79627190, chr5:158532400-158532520, chr2:131792932-131793052, chr3:194208336-194208456, chr17:46673964-46674084, chr4:147560771-147561088, chr12:133464324-133464463, chr19:58038983-58039194 and chr3:157812609-157812815.

[0020] Furthermore, the differentially methylated regions also include: at least one of chr19:56915342-56915462, chr7:37488070-37488477, chr19:56904581-56904938, chr17:37321401-37321702, chr7:87229906-87230345, chr8:99986819-99986939, chr2:176992936-176993235, chr7:37960353-37960473 and chr6:21667550-21667670.

[0021] Furthermore, the differentially methylated regions also include: at least one of chr10:97804121-97804338, chr20:61560374-61560535, chr6:78173165-78173373, chr1:8277528-8277747, chr17:29298072-29298242, chr7:27196138-27196564, chr18:67068779-67069168, chr3:69591929-69592195, chr3:59035661-59035943 and chr15:41805632-41805752.

[0022] Furthermore, the differentially methylated regions also include: at least one of chr2:63285358-63285536, chr4:96471066-96471186, chr20:23029625-23030082, chr1:243646395-243646735, chr16:68544226-68544346, chr5:35925138-35925258, chr11:44331133-44331538, chr10:20105505-20105717, chr1:18971873-18972006 and chr6:1384523-1384807.

[0023] In some specific embodiments, the differentially methylated region is selected from any one of the following combinations or any combination thereof:

[0024] 2marker group:

[0025] chr17:49021947-49022096, chr19:11998652-11998772;

[0026] 6marker group:

[0027] chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813 , chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960;

[0028] 8marker group:

[0029] chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058;

[0030] 10marker group:

[0031] chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895 , chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35757267-35757387, chr11:86383212-86383675;

[0032] 12marker group:

[0033] chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813 , chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35757267-35757387, chr11:86383212-86383675, chr12:22094867-22095025, chr3:99594764-99594983;

[0034] 17marker group:

[0035] chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19: 11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35757267 -35757387, chr11:86383212-86383675, chr12:22094867-22095025, chr3:99594764-99594983, chr14:60386224-6038 6387, chr17:36103848-36104221, chr17:59539346-59539760, chr14:60097125-60097295, chr19:56989290-56989474;

[0036] 22marker group:

[0037] chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-374 87813, chr7:64349109-64349229, chr19:11998775-11998895, chr6:2830384 0-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35 757267-35757387, chr11:86383212-86383675, chr12:22094867-22095025, c hr3:99594764-99594983, chr14:60386224-60386387, chr17:36103848-3610 4221, chr17:59539346-59539760, chr14:60097125-60097295, chr19:569892 90-56989474、chr1:200011526-200011646、chr12:54383600-54383720、chr1 7:7348591-7348915, chr17:36103471-36103693, chr10:94831568-94832005;

[0038] 30 marker group:

[0039] chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-6434 9229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938- 60386058, chr9:35757267-35757387, chr11:86383212-86383675, chr12:22094867-22095025, chr3:99594 764-99594983、chr14:60386224-60386387、chr17:36103848-36104221、chr17:59539346-59539760、chr14 :60097125-60097295, chr19:56989290-56989474, chr1:200011526-200011646, chr12:54383600-5438372 0. chr17:7348591-7348915, chr17:36103471-36103693, chr9:79627070-79627190, chr5:158532400-1585 32520, chr2:131792932-131793052, chr3:194208336-194208456, chr17:46673964-46674084, chr4:14756 0771-147561088, chr12:133464324-133464463, chr19:58038983-58039194, chr3:157812609-157812815;

[0040] 40marker group:

[0041] chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, c hr6:28303840-28303960、chr7:87229481-87229905、chr14:60385938-60386058、chr9:35757267-35757387、chr11:86383212-86383675、chr 12:22094867-22095025, chr3:99594764-99594983, chr14:60386224-60386387, chr17:36103848-36104221, chr17:59539346-59539760, chr 14:60097125-60097295, chr19:56989290-56989474, chr1:200011526-200011646, chr12:54383600-54383720, chr17:7348591-7348915, chr1 7:36103471-36103693, chr10:94831568-94832005, chr19:56915342-56915462, chr7:37488070-37488477, chr19:56904581-56904938, chr1 7:37321401-37321702, chr7:87229906-87230345, chr8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-79627190, chr5:1 c hr12:133464324-133464463, chr19:58038983-58039194, chr3:157812609-157812815, chr7:37960353-37960473, chr6:21667550-21667670;

[0042] 50marker group:

[0043] <h2 style=";text-align:left;direction:ltr">chr17:49021947-49022096,chr19:11998652-11998772,chr7:37487534-37487813,chr7:64349109-64349229,chr19:11998775-11998895,chr6 :28303840-28303960、chr7:87229481-87229905、chr14:60385938-6038 6058、chr9:35757267-35757387、chr11:86383212-86383675、chr12:220 94867-22095025、chr3:99594764-99594983、chr14:60386224-60386387 、chr17:36103848-36104221、chr17:59539346-59539760、chr14:600971 25-60097295、chr19:56989290-56989474、chr1:200011526-200011646、 chr12:54383600-54383720、chr17:7348591-7348915、chr17:36103471-3 6103693,chr10:94831568-94832005,chr19:56915342-56915462,chr7:37488070-37488477,chr19:56904581-56904938,chr17:37321401-3732 1702, chr7:87229906-87230345, chr8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-79627190, chr5:158532400-158532520 、chr2:131792932-131793052、chr3:194208336-194208456、chr17:4667 3964-46674084、chr4:147560771-147561088、chr12:133464324-133464 463, chr19:58038983-58039194, chr3:157812609-157812815, chr7:37960353-37960473, chr6:21667550-21667670, chr10:97804121-97804338chr20:61560374-61560535、chr6:78173165-78173373、chr1:8277528-8277747、chr17:29298072-29298242、chr7:27196138-27196564、chr18:67068779-67069168、chr3:69591929-69592195、chr3:59035661-59035943、chr15:41805632-41805752;、

[0044] 60marker:

[0045] <h2 style=";text-align:left;direction:ltr">chr17:49021947-49022096,chr19:11998652-11998772,chr7:37487534-37487813,chr7:64349109-64349229,chr19:11998775-11998895,chr6 :28303840-28303960、chr7:87229481-87229905、chr14:60385938-6038 6058、chr9:35757267-35757387、chr11:86383212-86383675、chr12:220 94867-22095025、chr3:99594764-99594983、chr14:60386224-60386387 、chr17:36103848-36104221、chr17:59539346-59539760、chr14:600971 25-60097295、chr19:56989290-56989474、chr1:200011526-200011646、 chr12:54383600-54383720、chr17:7348591-7348915、chr17:36103471-3 6103693,chr10:94831568-94832005,chr19:56915342-56915462,chr7:37488070-37488477,chr19:56904581-56904938,chr17:37321401-3732 1702, chr7:87229906-87230345, chr8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-79627190, chr5:158532400-158532520 、chr2:131792932-131793052、chr3:194208336-194208456、chr17:4667 3964-46674084、chr4:147560771-147561088、chr12:133464324-133464 463, chr19:58038983-58039194, chr3:157812609-157812815, chr7:37960353-37960473, chr6:21667550-21667670, chr10:97804121-97804338chr20:61560374-61560535, chr6:78173165-78173373, chr1:8277528-8277747, chr17:29298072-29298242, chr7:27196138-2719 6564, chr18:67068779-67069168, chr3:69591929-69592195, chr3:59035661-59035943, chr15:41805632-41805752, chr2:6328535 8-63285536, chr4:96471066-96471186, chr20:23029625-23030082, chr1:243646395-243646735, chr16:68544226-68544346, chr5 :35925138-35925258, chr11:44331133-44331538, chr10:20105505-20105717, chr1:18971873-18972006, chr6:1384523-1384807. ,

[0046] In some embodiments, the esophageal cancer is esophageal cancer present in a subject; optionally, the subject is a mammal; preferably, the mammal is a human.

[0047] In some embodiments, the esophageal cancer is selected from stage I, stage II, stage III, or stage IV esophageal cancer.

[0048] In some embodiments, a difference between the methylation level of the methylation biomarker in the test sample and the methylation level of the methylation biomarker in a sample from a subject without esophageal cancer indicates that the subject corresponding to the test sample has esophageal cancer.

[0049] In some embodiments, the subject not suffering from esophageal cancer includes a healthy subject or a subject suffering from a benign disease of the digestive system.

[0050] In some embodiments, the sample to be tested is selected from one or more of tissue, whole blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant.

[0051] The second aspect of the present invention provides any one of the following uses (i) to (ii):

[0052] (i) Use of the methylation biomarker according to the first aspect of the present invention in the preparation of a reagent or kit for diagnosing esophageal cancer;

[0053] (ii) Use of a reagent for determining the methylation level of the methylation biomarker according to the first aspect of the present invention in the preparation of a reagent or a kit for diagnosing esophageal cancer.

[0054] In some embodiments, the esophageal cancer is esophageal cancer present in a subject; optionally, the subject is a mammal; preferably, the mammal is a human.

[0055] In some embodiments, the esophageal cancer is selected from stage I, stage II, stage III, or stage IV esophageal cancer.

[0056] In some embodiments, a difference between the methylation level of the methylation biomarker in the test sample and the methylation level of the methylation biomarker in a sample from a subject without esophageal cancer indicates that the subject corresponding to the test sample has esophageal cancer.

[0057] In some embodiments, the subject not suffering from esophageal cancer includes a healthy subject or a subject suffering from a benign disease of the digestive system.

[0058] In some embodiments, the sample to be tested is selected from one or more of tissue, whole blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant.

[0059] The third aspect of the present invention provides the methylation biomarker as described in the first aspect of the present invention, which is used for diagnosing esophageal cancer.

[0060] In some embodiments, the esophageal cancer is esophageal cancer present in a subject; optionally, the subject is a mammal; preferably, the mammal is a human.

[0061] In some embodiments, the esophageal cancer is selected from stage I, stage II, stage III, or stage IV esophageal cancer.

[0062] In some embodiments, a difference between the methylation level of the methylation biomarker in the test sample and the methylation level of the methylation biomarker in a sample from a subject without esophageal cancer indicates that the subject corresponding to the test sample has esophageal cancer.

[0063] In some embodiments, the subject not suffering from esophageal cancer includes a healthy subject or a subject suffering from a benign disease of the digestive system.

[0064] In some embodiments, the sample to be tested is selected from one or more of tissue, whole blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant.

[0065] A fourth aspect of the present invention provides a method for diagnosing esophageal cancer, comprising the following steps:

[0066] Obtaining a sample to be tested from a subject;

[0067] Extracting genomic DNA and / or free DNA from the sample to be tested;

[0068] Detecting the methylation level of the methylation biomarker as described in the first aspect of the present invention in the DNA;

[0069] Determine whether the subject has esophageal cancer.

[0070] In some embodiments, the esophageal cancer is esophageal cancer present in a subject; optionally, the subject is a mammal; preferably, the mammal is a human.

[0071] In some embodiments, the esophageal cancer is selected from stage I, stage II, stage III, or stage IV esophageal cancer.

[0072] In some embodiments, a difference between the methylation level of the methylation biomarker in the test sample and the methylation level of the methylation biomarker in a sample from a subject without esophageal cancer indicates that the subject corresponding to the test sample has esophageal cancer.

[0073] In some embodiments, the subject not suffering from esophageal cancer includes a healthy subject or a subject suffering from a benign disease of the digestive system.

[0074] In some embodiments, the sample to be tested is selected from one or more of tissue, whole blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant.

[0075] In a fifth aspect, the present invention provides a kit for diagnosing esophageal cancer, wherein the kit comprises a reagent for detecting the methylation level of the methylation biomarker described in the first aspect of the present invention in a sample to be tested.

[0076] In some embodiments, the esophageal cancer is esophageal cancer present in a subject; optionally, the subject is a mammal; preferably, the mammal is a human.

[0077] In some embodiments, the esophageal cancer is selected from stage I, stage II, stage III, or stage IV esophageal cancer.

[0078] In some embodiments, a difference between the methylation level of the methylation biomarker in the test sample and the methylation level of the methylation biomarker in a sample from a subject without esophageal cancer indicates that the subject corresponding to the test sample has esophageal cancer.

[0079] In some embodiments, the subject not suffering from esophageal cancer includes a healthy subject or a subject suffering from a benign disease of the digestive system.

[0080] In some embodiments, the sample to be tested is selected from one or more of tissue, whole blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant.

[0081] In some embodiments, the reagent is a reagent used in a method for detecting methylation levels selected from the following: fluorescent quantitative PCR, methylation-specific PCR, digital PCR, DNA methylation chip, targeted DNA methylation sequencing, whole genome methylation sequencing, and one or more of DNA methylation mass spectrometry.

[0082] A sixth aspect of the present invention provides a system for diagnosing esophageal cancer, wherein the system comprises a detection device, a calculation device and an output device;

[0083] The detection device comprises an injector and a detector, wherein the injector is used to collect a sample to be tested from a subject, and the detector is used to detect the methylation level of the methylation biomarker described in the first aspect of the present invention in the sample to be tested;

[0084] The computing device includes a memory and a processor, wherein the memory stores a computer program, and the processor is configured to execute the computer program stored in the memory to achieve the following determination:

[0085] If the methylation level of the methylation biomarker described in the first aspect of the present invention in the sample is different from the methylation level of the methylation biomarker measured in a sample from a subject not suffering from esophageal cancer, it is determined that the subject corresponding to the sample has esophageal cancer.

[0086] In some embodiments, the esophageal cancer is esophageal cancer present in a subject; optionally, the subject is a mammal; preferably, the mammal is a human.

[0087] In some embodiments, the esophageal cancer is selected from stage I, stage II, stage III, or stage IV esophageal cancer.

[0088] In some embodiments, the subject not suffering from esophageal cancer includes a healthy subject or a subject suffering from a benign disease of the digestive system.

[0089] In some embodiments, the sample to be tested is selected from one or more of tissue, whole blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 : Methylation heatmap of 60 markers in 525 samples.

[0091] Figure 2 : Flow chart of the biomarker study described in the present invention.

[0092] Figure 3 : Schematic diagram of ROC curves of 2, 6, 8, 10, 12, 17, 22, 30, 40, 50, and 60 marker models (corresponding to 2marker group, 6marker group, 8marker group, 10marker group, 12marker group, 17marker group, 22marker group, 30marker group, 40marker group, 50marker group, and 60marker group, respectively) in the training set, validation set, and independent validation set samples. DETAILED DESCRIPTION

[0093] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0094] The experimental methods in the following examples of the present invention, for which specific conditions are not specified, are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0095] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0096] To facilitate understanding of the present technology, certain terms and phrases are defined below.

[0097] In this specification, the term "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the related objects are in an "or" relationship.

[0098] In this specification, the term "cancer" (also called carcinoma) generally refers to any type of malignant neoplasm, i.e., any morphological and / or physiological changes (based on genetic re-programming) in target cells that show or have a tendency to develop cancerous characteristics compared to unaffected (healthy) wild-type control cells. Examples of such changes may involve cell size and shape (becoming larger or smaller), cell proliferation (increase in cell number), cell differentiation (change in physiological state), apoptosis (programmed cell death) or cell survival. Therefore, the term "esophageal cancer" refers to cancerous growths in the parenchyma of the esophagus.

[0099] In this specification, the term "nucleic acid detection" refers to any method for determining the nucleotide composition of a target nucleic acid. Nucleic acid detection assays include, but are not limited to, DNA sequencing methods and probe hybridization methods.

[0100] In this specification, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, wherein the methyl moiety is not present in a recognized typical nucleotide base. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at the 5th position of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide, and 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at the 5th position of its pyrimidine ring. However, for the purposes of this article, thymine is not considered to be a methylated nucleotide when present in DNA, because thymine is a typical nucleotide base of DNA.

[0101] In this specification, "methylation level", "methylation degree", "methylation state", "methylation profile" and "methylation status" of a nucleic acid molecule refer to the presence or absence of one or more methylated nucleotide bases in a nucleic acid molecule. For example, a nucleic acid molecule containing methylated cytosine is considered to be methylated (e.g., the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered to be unmethylated.

[0102] In this specification, the methylation state can be optionally represented or indicated by a "methylation value" (e.g., representing the methylation frequency, score, ratio, percentage, etc.). The methylation value can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing the amplification spectrum after a bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated nucleic acids. Therefore, values ​​such as methylation values ​​represent the methylation state and can therefore be used as a quantitative indicator of the methylation state in multiple copies of a locus. The degree of co-methylation is represented or indicated by the methylation state of more than one methylation site, and co-methylation is defined as when the methylation state of more than one methylation site is methylated within a methylated region.

[0103] In this specification, "methylation rate", "methylation frequency" or "methylation percentage (%)" refers to the number of instances where a molecule or locus is methylated relative to the number of instances where the molecule or locus is unmethylated. For example, in some embodiments, the methylation rate of each target region is calculated as the number of co-methylated reads / total number of reads, where co-methylated reads are defined as reads in which 3 or more CpG sites are C in a window of 5 consecutive CpG sites in the target region.

[0104] In this specification, when the sequences differ in the degree (e.g., one has increased or decreased methylation relative to the other), frequency or pattern of methylation, the sequences are referred to as "differentially methylated" or as having a "methylation difference" or having a "different methylation state". The term "differential methylation" refers to the difference in the level or pattern of nucleic acid methylation in a cancer-positive sample compared to the level or pattern of nucleic acid methylation in a cancer-negative sample.

[0105] In this specification, the term "differentially methylated region" (DMR) refers to a DNA region containing one or more differentially methylated sites.

[0106] In the present specification, the term "methylation assay" or "methylation level detection" or "methylation degree detection" refers to any assay for determining the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0107] In this specification, the term "bisulfite reagent" refers to a reagent that, in some embodiments, comprises bisulfite, disulfite, hydrogen sulfite or a combination thereof. After DNA is treated with a bisulfite reagent, unmethylated cytosine nucleotides are converted into uracil, while methylated cytosine and other bases remain unchanged, thereby making it possible to distinguish between methylated and unmethylated cytidine in, for example, a CpG dinucleotide sequence.

[0108] In this specification, the term "sample" refers to any substance that may contain a target molecule that needs to be analyzed, including biological samples. As used herein, "sample" or "biological sample" refers to any sample obtained from a live or viral (or prion) source or other macromolecule and biomolecule source, and includes any cell type or tissue of a subject from which nucleic acids, proteins and / or other macromolecules can be obtained. Samples or biological samples can be samples obtained directly from biological sources or processed samples. Samples or biological samples include, but are not limited to, body fluids (such as whole blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, sweat, semen, feces, sputum, tears, mucus, amniotic fluid, etc.), exudates, bone marrow samples, ascites, pelvic washings, pleural fluid, spinal fluid, lymph, eye fluid, extracts from nasal, throat or genital swabs, cell suspensions of digested tissues, or extracts of fecal matter, and tissue and organ samples from humans, animals (such as non-human mammals) and plants, and processed samples derived therefrom.

[0109] In this specification, the term "subject" can be a mammal or a cell, tissue, organ or part of the mammal. In the present invention, mammal refers to any kind of mammal, preferably a human (including a human, a human subject or a human patient). Subjects and mammals include, but are not limited to, farm animals, sports animals, pets, primates, horses, dogs, cats and rodents such as mice and rats.

[0110] In this specification, the term "extracellular DNA" or its synonyms "cfDNA (circulating free DNA)", "circulating DNA" and "free circulating DNA" refers to DNA that is not contained in intact cells in the sample or the corresponding body fluid from which the sample is obtained, but is freely circulating in the body fluid sample. Extracellular DNA is usually fragmented genomic DNA.

[0111] As used in the present invention, the term "sensitivity" refers to the proportion of positive samples detected from confirmed positive samples, and its calculation formula is: sensitivity = (detected positive / true positive), and true positive is confirmed as positive using the recognized gold standard. "Specificity" refers to the proportion of normal samples detected from a certain normal person, and its calculation formula is specificity = (detected negative / true negative).

[0112] In this specification, 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 graph of the true positive ratio relative to the false positive ratio for different possible block cut points of a diagnostic test. It shows a compromise between sensitivity and specificity according to the selected 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 the accuracy of a diagnostic test (the larger the area, the better; the best is 1; a random test will have an ROC curve on the diagonal with an area of ​​0.5; see: JPEgan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0113] As used herein, diagnosis includes the detection or identification of a disease state or condition in a subject, determining the likelihood that a subject will develop a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis of a subject with a disease or condition (or its likely progression or regression), and determining the effectiveness of a treatment on a subject with a disease or condition.

[0114] The technical solution of the present invention is described in detail below.

[0115] The present invention provides a methylation biomarker associated with esophageal cancer, wherein the methylation biomarker comprises any one or any combination selected from the following differentially methylated regions:

[0116] <h2 style=";text-align:left;direction:ltr">chr17:49021947-49022096,chr19:11998652-11998772,chr7:37487534-37487813,chr7:64349109-64349229,chr19:11998775-11998895,chr6 :28303840-28303960、chr7:87229481-87229905、chr14:60385938-6038 6058、chr9:35757267-35757387、chr11:86383212-86383675、chr12:220 94867-22095025、chr3:99594764-99594983、chr14:60386224-60386387 、chr17:36103848-36104221、chr17:59539346-59539760、chr14:600971 25-60097295、chr19:56989290-56989474、chr1:200011526-200011646、 chr12:54383600-54383720、chr17:7348591-7348915、chr17:36103471-3 6103693,chr10:94831568-94832005,chr19:56915342-56915462,chr7:37488070-37488477,chr19:56904581-56904938,chr17:37321401-3732 1702, chr7:87229906-87230345, chr8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-79627190, chr5:158532400-158532520 、chr2:131792932-131793052、chr3:194208336-194208456、chr17:4667 3964-46674084、chr4:147560771-147561088、chr12:133464324-133464 463, chr19:58038983-58039194, chr3:157812609-157812815, chr7:37960353-37960473, chr6:21667550-21667670, chr10:97804121-97804338chr20:61560374-61560535, chr6:78173165-78173373, chr1:8277528-8277747, chr17:29298072-29298242, chr7:27196138-2719 6564, chr18:67068779-67069168, chr3:69591929-69592195, chr3:59035661-59035943, chr15:41805632-41805752, chr2:6328535 8-63285536, chr4:96471066-96471186, chr20:23029625-23030082, chr1:243646395-243646735, chr16:68544226-68544346, chr5 :35925138-35925258, chr11:44331133-44331538, chr10:20105505-20105717, chr1:18971873-18972006, chr6:1384523-1384807. ,

[0117] In some embodiments, the methylation biomarkers associated with esophageal cancer include at least one of the following differentially methylated regions: chr17:49021947-49022096, chr19:11998652-11998772.

[0118] In some embodiments, the biomarkers associated with esophageal cancer include at least one of the following differentially methylated regions: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960.

[0119] In some embodiments, the methylation biomarkers associated with esophageal cancer include at least one of the following differentially methylated regions: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058.

[0120] In some embodiments, the methylation biomarkers associated with esophageal cancer include at least one of the following differentially methylated regions: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr r19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, c hr14:60385938-60386058, chr9:35757267-35757387, chr11:86383212-86383675.

[0121] In some embodiments, the methylation biomarkers associated with esophageal cancer include at least one of the following differentially methylated regions: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, chr r6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35757267 -35757387, chr11:86383212-86383675, chr12:22094867-22095025, chr3:99594764-99594983.

[0122] In some embodiments, the methylation biomarkers associated with esophageal cancer include at least one of the following differentially methylated regions: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-64349229 0386058, chr9:35757267-35757387, chr11:86383212-86383675, chr12:22094867-22095025, chr3:99594764-99594983, chr14:603 86224-60386387, chr17:36103848-36104221, chr17:59539346-59539760, chr14:60097125-60097295, chr19:56989290-56989474.

[0123] In some embodiments, the methylation biomarkers associated with esophageal cancer include at least one of the following differentially methylated regions: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr1 9:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr1 4:60385938-60386058, chr9:35757267-35757387, chr11:86383212-86383675, chr1 2:22094867-22095025, chr3:99594764-99594983, chr14:60386224-60386387, chr 17:36103848-36104221, chr17:59539346-59539760, chr14:60097125-60097295, ch r19:56989290-56989474, chr1:200011526-200011646, chr12:54383600-54383720 , chr17:7348591-7348915, chr17:36103471-36103693, chr10:94831568-94832005.

[0124] In some embodiments, the methylation biomarkers associated with esophageal cancer include at least one of the following differentially methylated regions: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87 229481-87229905, chr14:60385938-60386058, chr9:35757267-35757387, chr11:86383212-86383675, chr12:22 094867-22095025, chr3:99594764-99594983, chr14:60386224-60386387, chr17:36103848-36104221, chr17:59 539346-59539760, chr14:60097125-60097295, chr19:56989290-56989474, chr1:200011526-200011646, chr12: 54383600-54383720, chr17:7348591-7348915, chr17:36103471-36103693, chr9:79627070-79627190, chr5:158 532400-158532520, chr2:131792932-131793052, chr3:194208336-194208456, chr17:46673964-46674084, chr4 :147560771-147561088, chr12:133464324-133464463, chr19:58038983-58039194, chr3:157812609-157812815.

[0125] In some embodiments, the methylation biomarkers associated with esophageal cancer include at least one of the following differentially methylated regions: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35757267-35757 387, chr11:86383212-86383675, chr12:22094867-22095025, chr3:99594 764-99594983, chr14:60386224-60386387, chr17:36103848-36104221, c hr17:59539346-59539760, chr14:60097125-60097295, chr19:56989290- 56989474, chr1:200011526-200011646, chr12:54383600-54383720, chr17 :7348591-7348915、chr17:36103471-36103693、chr10:94831568-948320 05. chr19:56915342-56915462, chr7:37488070-37488477, chr19:569045 81-56904938、chr17:37321401-37321702、chr7:87229906-87230345、chr 8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-7962 7190, chr5:158532400-158532520, chr2:131792932-131793052, chr3:19 4208336-194208456, chr17:46673964-46674084, chr4:147560771-147561 088、chr12:133464324-133464463、chr19:58038983-58039194、chr3:157 812609-157812815, chr7:37960353-37960473, chr6:21667550-21667670.

[0126] In some embodiments, the methylation biomarkers associated with esophageal cancer include at least one of the following differentially methylated regions: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35757267-35757 387, chr11:86383212-86383675, chr12:22094867-22095025, chr3:99594 764-99594983, chr14:60386224-60386387, chr17:36103848-36104221, c hr17:59539346-59539760, chr14:60097125-60097295, chr19:56989290- 56989474, chr1:200011526-200011646, chr12:54383600-54383720, chr17 :7348591-7348915、chr17:36103471-36103693、chr10:94831568-948320 05. chr19:56915342-56915462, chr7:37488070-37488477, chr19:569045 81-56904938、chr17:37321401-37321702、chr7:87229906-87230345、chr 8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-7962 7190, chr5:158532400-158532520, chr2:131792932-131793052, chr3:19 4208336-194208456, chr17:46673964-46674084, chr4:147560771-147561 088、chr12:133464324-133464463、chr19:58038983-58039194、chr3:157 812609-157812815、chr7:37960353-37960473、chr6:21667550-21667670、chr10:97804121-97804338、chr20:61560374-61560535、chr6:78173165-78173373、chr1:8277528-8277747、chr17:29298072-29298242、chr7:27196138-27196564、chr18:67068779-67069168、chr3:69591929-69592195、chr3:59035661-59035943、chr15:41805632-41805752。、

[0127] In other embodiments, the methylation biomarkers associated with esophageal cancer include at least one of the following differentially methylated regions: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35757267-3575 7387, chr11:86383212-86383675, chr12:22094867-22095025, chr3:9959 4764-99594983, chr14:60386224-60386387, chr17:36103848-36104221, c hr17:59539346-59539760, chr14:60097125-60097295, chr19:56989290- 56989474, chr1:200011526-200011646, chr12:54383600-54383720, chr17 :7348591-7348915、chr17:36103471-36103693、chr10:94831568-948320 05. chr19:56915342-56915462, chr7:37488070-37488477, chr19:569045 81-56904938、chr17:37321401-37321702、chr7:87229906-87230345、chr 8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-7962 7190, chr5:158532400-158532520, chr2:131792932-131793052, chr3:19 4208336-194208456, chr17:46673964-46674084, chr4:147560771-147561 088、chr12:133464324-133464463、chr19:58038983-58039194、chr3:157 812609-157812815、chr7:37960353-37960473、chr6:21667550-21667670、chr10:97804121-97804338, chr20:61560374-61560535, chr6:78173165-78173373, chr1:8277528-8277747, chr17:29298072-29298242, chr7:27196138-27196564, chr18:67068779-67069168, chr3:69591929-69592195, chr3:59035661-59035943, chr15:41805632-41805752, chr2:63285358-63285536, chr4:96471066-96471186, chr20:23029625-23030082, chr1:243646395-243646735, chr16:68544226-6854434 6. chr5:35925138-35925258, chr11:44331133-44331538, chr10:20105505-20105717, chr1:18971873-18972006, chr6:1384523-1384807. ,

[0128] Those skilled in the art understand that the methylation biomarker may also include a continuous segment of at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the full-length sequence of each differentially methylated region. In some embodiments, the continuous segment contains part or all of the (differential) methylation sites in the original differentially methylated region. For the embodiments of the methylation biomarker described above, the same applies to continuous segments from differentially methylated regions. In some embodiments, a continuous segment of a differentially methylated region and another differentially methylated region may also be used in combination to constitute the embodiments of the methylation biomarker described above.

[0129] Another object of the present invention is to provide the use of the above-mentioned methylation biomarkers, and / or reagents for detecting the methylation level thereof in the preparation of a detection reagent or kit for esophageal cancer. In some specific embodiments, the use of the above-mentioned methylation biomarkers in the preparation of a reagent or kit for diagnosing esophageal cancer is provided, and in other specific embodiments, the use of reagents for determining the methylation level of the above-mentioned methylation biomarkers in the preparation of a reagent or kit for diagnosing esophageal cancer is provided.

[0130] Another object of the present invention is to provide the above methylation biomarker for use in diagnosing esophageal cancer.

[0131] Another object of the present invention is to provide a kit for detecting esophageal cancer, comprising a reagent for detecting the above-mentioned methylation biomarker. In some specific embodiments, a kit for diagnosing esophageal cancer is provided, wherein the kit comprises a reagent for detecting the methylation level of the above-mentioned methylation biomarker in a sample to be tested.

[0132] In some embodiments, the kit can be used for the following platforms: using fluorescent quantitative PCR (qPCR), methylation-specific PCR (mPCR), digital PCR (ddPCR), DNA methylation chip, targeted DNA methylation sequencing, whole gene methylation sequencing (WGBS), DNA methylation mass spectrometry (MassArray), or reagents used in combination thereof. In some preferred embodiments, the kit is used for targeted DNA methylation sequencing.

[0133] Another object of the present invention is to provide a method for diagnosing esophageal cancer, comprising the following steps:

[0134] Obtaining a sample to be tested from a subject;

[0135] Extracting genomic DNA and / or free DNA from the sample to be tested;

[0136] Detecting the methylation level of the above-mentioned methylation biomarker in the DNA;

[0137] Determine whether the subject has esophageal cancer.

[0138] In some specific embodiments, the method further comprises treating the extracted genomic DNA and / or free DNA of the sample to be tested with bisulfite to obtain converted DNA.

[0139] In some specific embodiments, in the step of detecting the methylation level of the above-mentioned methylation biomarker in the DNA, the detection is performed by using fluorescent quantitative PCR (qPCR), methylation-specific PCR (mPCR), digital PCR (ddPCR), DNA methylation chip, targeted DNA methylation sequencing, whole genome methylation sequencing (WGBS), DNA methylation mass spectrometry (MassArray). In some preferred embodiments, the detection is performed by using a targeted DNA methylation sequencing method.

[0140] In some specific embodiments, the sample to be detected is a biopsy sample, for example, the biopsy sample is tissue, blood, or body fluid.

[0141] In some preferred embodiments of the present invention, the sample to be tested is whole blood, plasma or serum. Due to the convenience of sampling and other aspects, the diagnosis of the present invention includes early screening, detection / diagnosis or auxiliary detection / diagnosis of esophageal cancer. In some specific embodiments of the present invention, the methylated region is a methylated region present in cfDNA. In some more specific embodiments, the methylated region is a methylated region present in cfDNA in whole blood, plasma or serum samples.

[0142] The present invention screened out suitable DNA methylation biomarkers for detecting esophageal cancer, and the DNA methylation biomarkers can achieve the purpose of accurate prediction. The new DNA methylation markers developed by the present invention can be used to identify early esophageal cancer, can assist in accurate clinical diagnosis and guide treatment, and have also begun to be used in laboratory technical research.

[0143] In some specific embodiments, the esophageal cancer is esophageal cancer present in a subject, which is a malignant tumor of different classifications, different tumor sizes and different stages. Optionally, the subject is a mammal; preferably, the mammal is a human.

[0144] In some specific embodiments, if the methylation level of the methylation biomarker in the test sample is different from the methylation level of the methylation biomarker in samples from healthy subjects or adjacent cancer tissues or subjects with other cancers (non-esophageal cancer) or benign diseases, it indicates that the subject corresponding to the test sample has esophageal cancer.

[0145] Example 1: Detection method of novel ctDNA methylation markers for diagnosis of esophageal cancer

[0146] The methylation library and sequencing were performed on the 525 blood samples collected. The process and steps are as follows:

[0147] 1.1 DNA extraction and methylation library construction

[0148] 1.1.1 DNA extraction

[0149] Using MagMAX TM Circulating free DNA extraction kit (MagMAX TM Cell-Free DNA Isolation Kit) was used to extract circulating free DNA in plasma. For specific steps, please refer to the product manual.

[0150] 1.1.2 Conversion

[0151] After DNA is treated with bisulfite conversion, the unmethylated C bases in the CG double-base sequence in the DNA double strand will be converted to U bases, while the C bases in the methylated CG double-base sequence will not undergo base conversion. The extracted plasma cfDNA was bisulfite converted using the DNA bisulfite conversion kit Zymo Lightning Conversion Reagent. For specific steps, please refer to the product manual.

[0152] 1.1.3 DNA methylation pre-library construction

[0153] After completing the bisulfite conversion treatment of plasma cfDNA samples, targeted DNA methylation pre-library construction was performed using the AnchorIRIS library construction kit.

[0154] 1.1.3.1, Reaction 1:

[0155] 1.1.3.1.1. Add the following reagents to the converted 17 μL sample for reaction:

[0156] Components Volume (μL) After conversion 17 Repair fluid 1 8 Repair fluid 2 5 Total volume 30

[0157] 1.1.3.1.2. Place the sample in a PCR instrument and perform the reaction according to the following procedure:

[0158] temperature time 20℃ 20min 65℃ 5min Heat cover 105℃

[0159] 1.1.3.1.3. When the second step of the PCR reaction (65°C) reaches 5 minutes, immediately remove the sample from the PCR instrument and directly insert it into ice for more than 2 minutes before proceeding to the next step.

[0160] 1.1.3.2, Reaction 2:

[0161] 1.1.3.2.1. Prepare the following reaction solution:

[0162]

[0163] 1.1.3.2.2. Place the sample in a PCR instrument and perform the reaction according to the following procedure:

[0164] temperature time 20℃ 30min 95℃ 5min 10℃ hold Heat cover 105℃

[0165] 1.1.3.3. Reaction 3: 1.1.3.3.1. Prepare the following reaction solution:

[0166]

[0167] 1.1.3.3.2. Place the sample in a PCR instrument and perform the reaction according to the following procedure:

[0168]

[0169] 1.1.3.3.3. XP Beads Purification I

[0170] Add 180 μL XP Beads (need to be equilibrated at room temperature for half an hour in advance) to purify the product of reaction 3, and elute with 31 μL EB. The specific steps of purification are as follows:

[0171] a. Take the reaction product of reaction 3 and centrifuge it. Add 180 μL of Agencourt AMPureBeads to each sample and mix well by pipetting.

[0172] b. Incubate at room temperature for 5 min.

[0173] c. Centrifuge immediately and place on a magnetic rack for 5 min. When the liquid becomes clear, discard the supernatant.

[0174] d. Add 500 μL of 80% anhydrous ethanol, let stand for 30 s, and aspirate away the ethanol.

[0175] e. Repeat step d) once.

[0176] f. Centrifuge briefly, place the PCR tube on a magnetic rack, and remove the remaining ethanol.

[0177] g. Open the lid and dry the magnetic beads for 2-3 minutes, being careful not to overdry them.

[0178] h. Add 31 μL EB for elution, mix thoroughly with a pipette, and let stand at room temperature for 3 min.

[0179] I. Centrifuge briefly, place the PCR tube on a magnetic rack, and let it stand for 3 min.

[0180] J. Pipette 30 μL of supernatant into a new 200 μL PCR tube.

[0181] 1.1.3.4, Reaction 4:

[0182] 1.1.3.4.1. Prepare the following reaction solution:

[0183]

[0184] 1.1.3.4.2. Place in PCR instrument and perform reaction according to the following procedure:

[0185] temperature time Number of cycles 37℃ 30min 1 95℃ 5min 1 10℃ Hold 1

[0186] 1.1.3.5, Reaction 5:

[0187] 1.1.3.5.1. Prepare the following reaction solution:

[0188] Components Volume (μL) Volume of product from the previous reaction (reaction 4) 50 Amplification Solution 1 48 Index primer mix 2 Total volume 100

[0189] 1.1.3.5.2. Place the sample in a PCR instrument and perform the reaction according to the following procedure:

[0190] 1.1.3.5.3. XP Beads Purification II

[0191] Add 71 μL XP Beads (need to be equilibrated at room temperature for half an hour in advance) to purify the PCR product of reaction 5, and elute with 55 μL EB. The specific steps of purification are as follows:

[0192] a. Take the reaction product of reaction 5 and centrifuge it. Add 71 μL XP Beads to each sample and mix well by pipetting. b. Incubate at room temperature for 5 min.

[0193] c. Centrifuge briefly and place on a magnetic rack for 5 min. After the liquid becomes clear, discard the supernatant.

[0194] d. Add 500 μL of 80% anhydrous ethanol, let stand for 30 s, and aspirate away the ethanol.

[0195] e. Repeat step d) once.

[0196] f. Centrifuge briefly, place the PCR tube on a magnetic rack, and remove the remaining ethanol.

[0197] g. Open the lid and dry the magnetic beads for 2-3 minutes, being careful not to overdry them.

[0198] h. Add 55 μL EB for elution, mix thoroughly with a pipette, and let stand at room temperature for 3 min.

[0199] i. Centrifuge briefly, place the PCR tube on a magnetic rack and let it stand for 3 min.

[0200] j. Pipette 54 μL of supernatant into a new 1.5 mL centrifuge tube.

[0201] k.Qubit quantification: Take 1 μL and use Qubit dsDNA HS Assay Kit to quantify the library.

[0202] 1.2. DNA methylation sequencing targeted enrichment

[0203] After the DNA methylation sequencing pre-library was constructed, the cfDNA pre-library (i.e. the purified reaction 5 product) was targeted enriched using the IDT xGen Lockdown Reagents kit. The specific steps are as follows:

[0204] 1.2.1. Prelibrary hybridization experimental steps

[0205] 1. Preparation:

[0206] 1. Calculate the input volume of each pre-library according to the concentration of the pre-library. The input volume of each pre-library is 250 ng. Every 8-16 pre-libraries can be placed in the same reaction tube (pool) for hybridization. When the number of pre-libraries is less than 8, other pre-libraries need to be used to make up.

[0207] 2. Thaw the probe solution at 4°C and thaw the other reagents at room temperature. After thawing, vortex and mix well for later use.

[0208] 3. Preheat the vacuum concentrator: set the temperature to 60°C, V-AQ mode, brake to OFF, and run for 20 min.

[0209] 4. Note that the amplification primer numbers of samples in the same reaction tube cannot be exactly the same.

[0210] 2. Specific process:

[0211] After all reagents and libraries are melted, they all need to be Vortex mixed and centrifuged for 5 seconds. The libraries are arranged in groups of four on the EP tube rack according to the hybridization table. xGen Hybridization Buffer Enhancer is kept at room temperature. All other reagents are placed on ice for later use.

[0212] 1. Add the pre-library into the corresponding 0.2 mL PCR tube according to the pre-library combination and input volume in the hybridization experiment record sheet.

[0213] 2. Add the components to prepare the premix as follows:

[0214]

[0215] Preparation for concentration reaction: Add 7 μL of premixed solution to the PCR tube in step 2. After adding and mixing, centrifuge instantly and concentrate according to the above reaction program settings until it is completely dried to powdery crystals. If there is more liquid, the drying time can be appropriately extended.

[0216] 3. Reaction 6:

[0217] Prepare the premix by adding the components on ice as follows and use immediately after preparation:

[0218]

[0219] PCR reaction preparation: Add 17 μL of premixed solution to the concentrated and dried PCR tube in step 2, mix the sample, centrifuge instantly, and perform the reaction according to the above PCR reaction program settings. Hybridize at 65℃ for 4-16h and then perform elution.

[0220] 1.2.2 Prelibrary elution experimental steps

[0221] 1. Preparation:

[0222] 1. Open the hybridization chamber in advance and set it to 65℃ for preheating.

[0223] 2. Take out the Dynabeads M-270 Streptavidin 30 minutes in advance and place it at room temperature for equilibrium. Put the remaining elution reagents into a thin film glove and thaw in the hybridization box until they are completely dissolved and free of crystals.

[0224] 3. Turn on the shaking thermostatic metal bath and set it to 65°C for preheating.

[0225] 4. Prepare 1× diluted working solution according to the number of reaction tubes (pool) as follows, and place the diluted working solution to be heated in a shaking constant temperature metal bath to preheat:

[0226]

[0227] 5.M270 magnetic beads preparation:

[0228] a. Vortex the M-270Beads thoroughly, take 50μL to a 1.5mL centrifuge tube, place it on a 1.5mL magnetic rack, let it stand for 30s, then aspirate and discard the supernatant to avoid aspirating the magnetic beads. A small amount of liquid may remain.

[0229] b. Take 100 μL of 1× Bead Wash Buffer into the centrifuge tube prepared in step a, vortex to mix, centrifuge briefly, place on a magnetic rack, and discard the supernatant after the liquid is clarified. Repeat the operation twice.

[0230] c. Prepare as follows: take 17 μL of the magnetic bead mixture into the centrifuge tube in step b, mix well by pipetting to resuspend the magnetic beads, and transfer to a 0.2 mL PCR tube for later use.

[0231]

[0232] 2. Experimental operation:

[0233] 1. Rapidly transfer the hybridized PCR tube to a 0.2mL 96-well yellow plate preheated in a 65℃ hybridization box, centrifuge quickly, and place in a 65℃ hybridization box after centrifugation. Transfer 17μL of the magnetic bead mixture containing M270 magnetic beads to the PCR tube of the hybridization pool sample, blow and mix, avoid bubbles during the operation, place it in the hybridization box, float on the float, start the rotor at medium speed, and rotate for 45 minutes or more. Repeat the operation for multiple samples.

[0234] 2. Prepare two 1.5 mL centrifuge tubes of the same quantity as the sample and number them. Preheat one tube in a 65°C metal bath and keep the other tube at room temperature for later use.

[0235] 3. Take 100μL of preheated xGen 1×Wash Buffer 1 diluent and add it to the sample. Mix well by pipetting. Transfer all to a preheated 1.5mL centrifuge tube. Centrifuge and place on a magnetic rack. After the liquid is clarified, discard the supernatant.

[0236] 4. Add 150 μL of preheated xGen 1× Stringent Wash Buffer, vortex to mix, incubate on a thermomixer at 65°C, 2000 rpm for 5 min, centrifuge, place on a magnetic rack, and discard the supernatant after the liquid is clarified. Repeat the operation once.

[0237] 5. Add 150 μL of xGen 1×Wash Buffer 1 diluent (without preheating), mix by pipetting, transfer to a new centrifuge tube, and incubate at room temperature at 1800 rpm for 2 minutes; centrifuge, place on a magnetic rack, and discard the supernatant after the liquid is clarified.

[0238] 6. Add 150 μL of xGen 1×Wash Buffer 2 diluent, vortex to mix, and incubate at room temperature at 1800 rpm for 2 minutes; centrifuge, place on a magnetic rack, and discard the supernatant after the liquid is clarified.

[0239] 7. Add 150 μL of xGen 1×Wash Buffer 3 diluent and vortex to mix. Mix at room temperature at 1800 rpm and incubate for 2 minutes. Centrifuge and place on a magnetic rack. After the liquid is clarified, discard the supernatant.

[0240] 8. Add 20 μL of DEPC water, vortex at medium speed to mix and resuspend the magnetic beads, and centrifuge for later use.

[0241] 9. Reaction 7:

[0242] Prepare a master mix by adding the following components on ice:

[0243]

[0244] PCR reaction preparation: Add the sample and mix as above, centrifuge briefly, place in PCR instrument and start the reaction program.

[0245] 10. Magnetic bead purification:

[0246] a.Equilibrate XP Beads at room temperature for 30 min, mix well, take 75 μL XP Beads and reaction product 7, incubate at room temperature for 5 min, centrifuge for 30 s, adsorb on magnetic rack for 5 min until the liquid is clear, aspirate and discard supernatant, rinse with 500 μL 80% ethanol for 30 s, discard supernatant, add 500 μL 80% ethanol to rinse for 30 s, discard supernatant, centrifuge for 1 min, put on the rack until the liquid is clear, aspirate the remaining liquid, open the lid and dry until the surface of the magnetic beads is non-reflective, add 22 μL EB to elute, mix well, incubate at room temperature for 5 min, centrifuge for 1-2 min, adsorb on the rack for 3-5 min until the liquid is clear, collect 20 μL supernatant.

[0247] b. The supernatant collected after purification is the final library. The concentration of the final library is measured using Qubit. Part of the final library is taken for sequencing, and the remaining final library samples are stored in a -20°C refrigerator.

[0248] 1.3. Use the sequencer of Illumina to sequence the samples after hybridization capture to obtain sequencing results.

[0249] 1.4 Analysis of Offboard Data

[0250] The raw fastq files obtained by sequencing were analyzed using fastp 0.19.6 software for the sequencing quality of each batch of data. If the proportion of Q30 bases in the batch data was higher than 75%, the quality control passed. The adapter sequences and low-quality base fragments introduced during the library construction process were removed from the sequencing reads to obtain clean fastq files. The clean reads were aligned to the human genome (hg19) using the sequence alignment software bismark v0.22.1 to obtain the alignment bam file. The quality control passed if the conversion efficiency of sulfite treatment was ≥97%. Reads were deduplicated according to UMI. After obtaining the deduplicated alignment bam file, the methylation status of CpG sites in the targeted target segment was extracted from the bam file. The methylation rate of each target region was calculated as the number of co-methylated reads / total number of reads. Among them, co-methylated reads are defined as reads with 3 or more CpG sites as C in the window of 5 consecutive CpG sites in the target region. The methylation rate was obtained for subsequent data analysis.

[0251] Example 2

[0252] This embodiment discloses a methylation-specific biomarker for diagnosing esophageal cancer. In the early stage, based on the methylation data of 186 esophageal cancer samples and 16 adjacent normal control samples from the cancer methylation public database TCGA and 37 esophageal cancer samples and 37 adjacent normal control samples from the local methylation EPIC sequencing database, after the methylation level difference test and analysis of different groups, combined with the literature and gene function annotations, a total of 60 methylation markers for diagnosing esophageal cancer were screened, and then a methylation targeted capture sequencing platform was established. A total of 525 blood samples were collected (115 esophageal cancer cases and 410 non-esophageal cancer cases), including 210 training set samples (47 esophageal cancer cases and 163 non-esophageal cancer cases), 157 validation set samples (34 esophageal cancer cases and 123 non-esophageal cancer cases) and 158 independent validation set samples (34 esophageal cancer cases and 124 non-esophageal cancer cases). Their clinical information is shown in Table 1. The 60 methylation markers (hereinafter referred to as markers) and the AUC values ​​of independent distinction are shown in Table 2. The heat map distribution is shown in Figure 1 .

[0253] Table 1: Clinical information of 525 blood samples

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267] In the above table, the staging of esophageal cancer adopts the UICC / AJCC staging (8th edition) standard.

[0268] Table 2. Specific performance data of 60 methylation markers in 525 blood samples of esophageal cancer and non-esophageal cancer patients

[0269]

[0270]

[0271]

[0272] Among them, chr7:64349109-64349229, chr19:11998775-11998895, and chr19:56904581-56904938 had the best overall performance, with AUC values ​​of 0.84.

[0273] Example 3

[0274] Utilize the 3 batches of data sets collected in Example 2, and then detect the methylation signals of 60 methylation markers of these samples according to the library construction and sequencing technology of Example 1, and use the two core methylation marker information (chr17:49021947-49022096, chr19:11998652-11998772). Take the methylation rate values ​​of the two methylation markers as input, and judge the negative / positive by the logistic regression formula fitting score method. The model calculation formula of the logistic regression obtained by the training set is as follows:

[0275] Logistic regression formula score = 1 / (1+e (-x) )

[0276] Where x = weight 1 × methylation marker 1 (e.g. chr17: 49021947-49022096) + weight 2 × methylation marker 2 (e.g. chr19: 11998652-11998772) + ... + weight n × methylation marker n + intercept, weight 1-n and intercept are constants. e represents a natural constant, and its value is approximately 2.718281828459045. Logistic regression formula fitting is implemented using the Logistic Regression software package of Sklearn, and the parameters are default parameters. The flowchart is shown in Figure 2 shown.

[0277] The model performance results for the two methylation markers showed that in the training set, the AUC was 84.26% ( Figure 3The sensitivity (SE) was 76.6%, the specificity (SP) was 79.75%, the positive predictive value (PPV) was 52.17%, the negative predictive value (NPV) was 92.2%, and the accuracy (ACC) reached 79.05%.

[0278] In the validation set, the AUC was 82.69% ( Figure 3 The sensitivity was SE of 73.53%, the specificity was SP of 76.42%, the positive predictive value PPV of 45.3%, the negative predictive value NPV of 91.26%, and the accuracy ACC of 75.8%.

[0279] In the independent validation set, the AUC was 87.77% ( Figure 3 The sensitivity was SE was 70.59%, the specificity was SP 83.87%, the positive predictive value PPV was 54.55%, the negative predictive value NPV was 91.23%, and the accuracy ACC reached 81.01%.

[0280] The ROC curves of each batch of data are shown in Figure 3 .

[0281] Example 4

[0282] Based on Example 3, the number of markers was randomly expanded to combinations of 6, 8, 10, 12, 17, 22, 30, 40, 50, and 60 markers, and then a logistic regression model was constructed for detection and verification. The performance of the training set is shown in Table 3, the performance of the verification set is shown in Table 4, and the performance of the independent verification set is shown in Table 5.

[0283] Table 3. Training set performance of each methylation marker combination in 525 esophageal cancer and benign control samples

[0284]

[0285] Table 4. Validation performance of each methylation marker combination in 525 esophageal cancer and benign control samples

[0286] AUC Sensitivity Specificity PPV NPV Accuracy Marker combination 0.86 0.82 0.83 0.57 0.94 0.83 6marker group 0.87 0.82 0.84 0.58 0.94 0.83 8marker group 0.88 0.82 0.84 0.58 0.94 0.83 10 marker group 0.88 0.76 0.86 0.60 0.93 0.84 12marker group 0.88 0.74 0.86 0.60 0.92 0.83 17marker group 0.89 0.79 0.86 0.61 0.94 0.85 22marker group 0.90 0.85 0.83 0.58 0.95 0.83 30 marker group 0.90 0.82 0.80 0.54 0.94 0.81 40marker group 0.90 0.88 0.81 0.57 0.96 0.83 50marker group 0.90 0.88 0.82 0.58 0.96 0.83 60marker group

[0287] Table 5. Performance of each methylation marker combination in an independent validation set of 525 esophageal cancer and benign control samples

[0288] AUC Sensitivity Specificity PPV NPV Accuracy Marker combination 0.90 0.79 0.83 0.56 0.94 0.82 6marker group 0.91 0.82 0.81 0.55 0.94 0.82 8marker group 0.91 0.82 0.81 0.54 0.94 0.81 10 marker group 0.91 0.79 0.90 0.68 0.94 0.87 12marker group 0.91 0.76 0.90 0.67 0.93 0.87 17marker group 0.91 0.76 0.88 0.63 0.93 0.85 22marker group 0.91 0.82 0.81 0.55 0.94 0.82 30 marker group 0.91 0.85 0.80 0.54 0.95 0.81 40marker group 0.93 0.88 0.81 0.57 0.96 0.83 50marker group 0.93 0.88 0.82 0.58 0.96 0.84 60marker group

[0289] From the above results, it can be seen that the AUC of the training set is above 0.86, the AUC of the validation set is above 0.86, and some combinations even reach 0.9. The AUC of the independent validation set is above 0.9 as a whole, indicating that the model constructed by the training set is relatively stable and these biomarkers also show good distinguishing performance in new samples, and can be used as methylation markers to distinguish esophageal cancer and non-esophageal cancer samples.

[0290] In summary, it can be seen that the methylation markers screened by this method have a very high correlation with the diagnosis of esophageal cancer.

[0291] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0292] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A methylation biomarker, wherein: The methylation biomarker includes any one or any combination of the following differentially methylated regions: <h2 style=";text-align:left;direction:ltr">chr17:49021947-49022096,chr19:11998652-11998772,chr7:37487534-37487813,chr7:64349109-64349229,chr19:11998775-11998895,chr6 :28303840-28303960、chr7:87229481-87229905、chr14:60385938-6038 6058、chr9:35757267-35757387、chr11:86383212-86383675、chr12:220 94867-22095025、chr3:99594764-99594983、chr14:60386224-60386387 、chr17:36103848-36104221、chr17:59539346-59539760、chr14:600971 25-60097295、chr19:56989290-56989474、chr1:200011526-200011646、 chr12:54383600-54383720、chr17:7348591-7348915、chr17:36103471-3 6103693,chr10:94831568-94832005,chr19:56915342-56915462,chr7:37488070-37488477,chr19:56904581-56904938,chr17:37321401-3732 1702, chr7:87229906-87230345, chr8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-79627190, chr5:158532400-158532520 、chr2:131792932-131793052、chr3:194208336-194208456、chr17:4667 3964-46674084、chr4:147560771-147561088、chr12:133464324-133464 463, chr19:58038983-58039194, chr3:157812609-157812815, chr7:37960353-37960473, chr6:21667550-21667670, chr10:97804121-97804338chr20:61560374-61560535、chr6:78173165-78173373、chr1:8277528-8277747、chr17:29298072-29298242、chr7:27196138-27196564、chr18:67068779-67069168、chr3:69591929-69592195、chr3:59035661-59035943、chr15:41805632-41805752、chr2:63285358-63285536、chr4:96471066-96471186、chr20:23029625-23030082、chr1:243646395-243646735、chr16:68544226-68544346、chr5:35925138-35925258、chr11:44331133-44331538、chr10:20105505-20105717、chr1:18971873-18972006、chr6:1384523-1384807。、 2. The methylation biomarker according to claim 1, wherein The differentially methylated region includes at least one of chr17:49021947-49022096 and chr19:11998652-11998772.

3. The methylation biomarker according to claim 2, wherein: The differentially methylated regions also include at least one of: chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895 and chr6:28303840-28303960.

4. The methylation biomarker according to claim 2 or 3, wherein: The differentially methylated region also includes at least one of chr7:87229481-87229905 and chr14:60385938-60386058.

5. The methylation biomarker according to any one of claims 2 to 4, wherein The differentially methylated region also includes at least one of chr9:35757267-35757387 and chr11:86383212-86383675.

6. The methylation biomarker according to any one of claims 2 to 5, wherein The differentially methylated region also includes at least one of chr12:22094867-22095025 and chr3:99594764-99594983.

7. The methylation biomarker according to any one of claims 2 to 6, wherein The differentially methylated regions also include at least one of: chr14:60386224-60386387, chr17:36103848-36104221, chr17:59539346-59539760, chr14:60097125-60097295 and chr19:56989290-56989474.

8. The methylation biomarker according to any one of claims 2 to 7, wherein The differentially methylated regions further include at least one of chr1:200011526-200011646, chr12:54383600-54383720, chr17:7348591-7348915 and chr17:36103471-36103693; Optionally, the differentially methylated region also includes chr10:94831568-94832005.

9. The methylation biomarker according to any one of claims 2 to 8, wherein The differentially methylated regions also include: at least one of chr9:79627070-79627190, chr5:158532400-158532520, chr2:131792932-131793052, chr3:194208336-194208456, chr17:46673964-46674084, chr4:147560771-147561088, chr12:133464324-133464463, chr19:58038983-58039194 and chr3:157812609-157812815.

10. The methylation biomarker according to any one of claims 2 to 9, wherein The differentially methylated regions also include: at least one of chr19:56915342-56915462, chr7:37488070-37488477, chr19:56904581-56904938, chr17:37321401-37321702, chr7:87229906-87230345, chr8:99986819-99986939, chr2:176992936-176993235, chr7:37960353-37960473 and chr6:21667550-21667670.

11. The methylation biomarker according to any one of claims 2 to 10, wherein The differentially methylated regions also include: at least one of chr10:97804121-97804338, chr20:61560374-61560535, chr6:78173165-78173373, chr1:8277528-8277747, chr17:29298072-29298242, chr7:27196138-27196564, chr18:67068779-67069168, chr3:69591929-69592195, chr3:59035661-59035943 and chr15:41805632-41805752.

12. The methylation biomarker according to any one of claims 2 to 11, wherein The differentially methylated regions also include: chr2:63285358-63285536, chr4:96471066-96471186, chr20:23029625-23030082, chr1:243646395-243646735, chr16:68544226-68544346, chr5:35925138-35925258, chr11:44331133-44331538, chr10:20105505-20105717, chr1:18971873-18972006 and at least one of chr6:1384523-1384807.

13. The methylation biomarker according to any one of claims 1 to 12, wherein The differentially methylated regions are selected from any one of the following combinations or any combination thereof: 2marker group: chr17:49021947-49022096, chr19:11998652-11998772; 6marker group: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813 , chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960; 8marker group: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058; 10marker group: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895 , chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35757267-35757387, chr11:86383212-86383675; 12marker group: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813 , chr7:64349109-64349229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35757267-35757387, chr11:86383212-86383675, chr12:22094867-22095025, chr3:99594764-99594983; 17 marker group: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19: 11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35757267 -35757387, chr11:86383212-86383675, chr12:22094867-22095025, chr3:99594764-99594983, chr14:60386224-6038 6387, chr17:36103848-36104221, chr17:59539346-59539760, chr14:60097125-60097295, chr19:56989290-56989474; 22marker group: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-374 87813, chr7:64349109-64349229, chr19:11998775-11998895, chr6:2830384 0-28303960, chr7:87229481-87229905, chr14:60385938-60386058, chr9:35 757267-35757387, chr11:86383212-86383675, chr12:22094867-22095025, c hr3:99594764-99594983, chr14:60386224-60386387, chr17:36103848-3610 4221, chr17:59539346-59539760, chr14:60097125-60097295, chr19:569892 90-56989474, chr1:200011526-200011646, chr12:54383600-54383720, chr1 7:7348591-7348915, chr17:36103471-36103693, chr10:94831568-94832005; 30 marker group: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-6434 9229, chr19:11998775-11998895, chr6:28303840-28303960, chr7:87229481-87229905, chr14:60385938- 60386058, chr9:35757267-35757387, chr11:86383212-86383675, chr12:22094867-22095025, chr3:99594 764-99594983、chr14:60386224-60386387、chr17:36103848-36104221、chr17:59539346-59539760、chr14 :60097125-60097295, chr19:56989290-56989474, chr1:200011526-200011646, chr12:54383600-5438372 0. chr17:7348591-7348915, chr17:36103471-36103693, chr9:79627070-79627190, chr5:158532400-1585 32520, chr2:131792932-131793052, chr3:194208336-194208456, chr17:46673964-46674084, chr4:14756 0771-147561088, chr12:133464324-133464463, chr19:58038983-58039194, chr3:157812609-157812815; 40marker group: chr17:49021947-49022096, chr19:11998652-11998772, chr7:37487534-37487813, chr7:64349109-64349229, chr19:11998775-11998895, c hr6:28303840-28303960、chr7:87229481-87229905、chr14:60385938-60386058、chr9:35757267-35757387、chr11:86383212-86383675、chr 12:22094867-22095025, chr3:99594764-99594983, chr14:60386224-60386387, chr17:36103848-36104221, chr17:59539346-59539760, chr 14:60097125-60097295, chr19:56989290-56989474, chr1:200011526-200011646, chr12:54383600-54383720, chr17:7348591-7348915, chr1 7:36103471-36103693, chr10:94831568-94832005, chr19:56915342-56915462, chr7:37488070-37488477, chr19:56904581-56904938, chr1 7:37321401-37321702, chr7:87229906-87230345, chr8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-79627190, chr5:1 c hr12:133464324-133464463, chr19:58038983-58039194, chr3:157812609-157812815, chr7:37960353-37960473, chr6:21667550-21667670; 50marker group: <h2 style=";text-align:left;direction:ltr">chr17:49021947-49022096,chr19:11998652-11998772,chr7:37487534-37487813,chr7:64349109-64349229,chr19:11998775-11998895,chr6 :28303840-28303960、chr7:87229481-87229905、chr14:60385938-6038 6058、chr9:35757267-35757387、chr11:86383212-86383675、chr12:220 94867-22095025、chr3:99594764-99594983、chr14:60386224-60386387 、chr17:36103848-36104221、chr17:59539346-59539760、chr14:600971 25-60097295、chr19:56989290-56989474、chr1:200011526-200011646、 chr12:54383600-54383720、chr17:7348591-7348915、chr17:36103471-3 6103693,chr10:94831568-94832005,chr19:56915342-56915462,chr7:37488070-37488477,chr19:56904581-56904938,chr17:37321401-3732 1702, chr7:87229906-87230345, chr8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-79627190, chr5:158532400-158532520 、chr2:131792932-131793052、chr3:194208336-194208456、chr17:4667 3964-46674084、chr4:147560771-147561088、chr12:133464324-133464 463, chr19:58038983-58039194, chr3:157812609-157812815, chr7:37960353-37960473, chr6:21667550-21667670, chr10:97804121-97804338chr20:61560374-61560535、chr6:78173165-78173373、chr1:8277528-8277747、chr17:29298072-29298242、chr7:27196138-27196564、chr18:67068779-67069168、chr3:69591929-69592195、chr3:59035661-59035943、chr15:41805632-41805752;、 60marker: <h2 style=";text-align:left;direction:ltr">chr17:49021947-49022096,chr19:11998652-11998772,chr7:37487534-37487813,chr7:64349109-64349229,chr19:11998775-11998895,chr6 :28303840-28303960、chr7:87229481-87229905、chr14:60385938-6038 6058、chr9:35757267-35757387、chr11:86383212-86383675、chr12:220 94867-22095025、chr3:99594764-99594983、chr14:60386224-60386387 、chr17:36103848-36104221、chr17:59539346-59539760、chr14:600971 25-60097295、chr19:56989290-56989474、chr1:200011526-200011646、 chr12:54383600-54383720、chr17:7348591-7348915、chr17:36103471-3 6103693,chr10:94831568-94832005,chr19:56915342-56915462,chr7:37488070-37488477,chr19:56904581-56904938,chr17:37321401-3732 1702, chr7:87229906-87230345, chr8:99986819-99986939, chr2:176992936-176993235, chr9:79627070-79627190, chr5:158532400-158532520 、chr2:131792932-131793052、chr3:194208336-194208456、chr17:4667 3964-46674084、chr4:147560771-147561088、chr12:133464324-133464 463, chr19:58038983-58039194, chr3:157812609-157812815, chr7:37960353-37960473, chr6:21667550-21667670, chr10:97804121-97804338chr20:61560374-61560535、chr6:78173165-78173373、chr1:8277528-8277747、chr17:29298072-29298242、chr7:27196138-27196564、chr18:67068779-67069168、chr3:69591929-69592195、chr3:59035661-59035943、chr15:41805632-41805752、chr2:63285358-63285536、chr4:96471066-96471186、chr20:23029625-23030082、chr1:243646395-243646735、chr16:68544226-68544346、chr5:35925138-35925258、chr11:44331133-44331538、chr10:20105505-20105717、chr1:18971873-18972006、chr6:1384523-1384807。、 14. The methylation biomarker according to any one of claims 1 to 13, wherein The esophageal cancer is esophageal cancer present in the subject; Optionally, the subject is a mammal; Preferably, the mammal is a human.

15. The methylation biomarker according to any one of claims 1 to 14, wherein The esophageal cancer is selected from stage I, stage II, stage III or stage IV esophageal cancer.

16. The methylation biomarker according to any one of claims 1 to 15, wherein If the methylation level of the methylation biomarker in the sample to be tested is different from the methylation level of the methylation biomarker in a sample from a subject who does not have esophageal cancer, it indicates that the subject corresponding to the sample to be tested has esophageal cancer; Optionally, the subject not suffering from esophageal cancer is a healthy subject or a subject suffering from a benign disease of the digestive system; Optionally, the sample to be tested is selected from one or more of tissue, whole blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant.

17. Any of the following uses (i) to (ii): (i) Use of the methylation biomarker according to any one of claims 1 to 16 in the preparation of a reagent or kit for diagnosing esophageal cancer; (ii) Use of a reagent for determining the methylation level of a methylation biomarker according to any one of claims 1 to 16 in the preparation of a reagent or kit for diagnosing esophageal cancer; Optionally, the esophageal cancer is esophageal cancer present in a subject; optionally, the subject is a mammal; preferably, the mammal is a human; Optionally, the esophageal cancer is selected from stage I, stage II, stage III or stage IV esophageal cancer; Optionally, if the methylation level of the methylation biomarker in the sample to be tested is different from the methylation level of the methylation biomarker in a sample from a subject not suffering from esophageal cancer, it indicates that the subject corresponding to the sample to be tested has esophageal cancer; Optionally, the subject not suffering from esophageal cancer includes a healthy subject or a subject suffering from a benign disease of the digestive system; Optionally, the sample to be tested is selected from one or more of tissue, whole blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant.

18. A kit for diagnosing esophageal cancer, wherein: The kit comprises a reagent for detecting the methylation level of the methylation biomarker according to any one of claims 1 to 16 in a sample to be tested; Optionally, the esophageal cancer is esophageal cancer present in a subject; optionally, the subject is a mammal; preferably, the mammal is a human; Optionally, the esophageal cancer is selected from stage I, stage II, stage III or stage IV esophageal cancer; Optionally, if the methylation level of the methylation biomarker in the sample to be tested is different from the methylation level of the methylation biomarker in a sample from a subject not suffering from esophageal cancer, it indicates that the subject corresponding to the sample to be tested has esophageal cancer; Optionally, the subject not suffering from esophageal cancer includes a healthy subject or a subject suffering from a benign disease of the digestive system; Optionally, the sample to be tested is selected from one or more of tissue, whole blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant.

19. The kit for diagnosing esophageal cancer according to claim 18, wherein: The reagent is selected from the following reagents used in the method for detecting methylation levels: one or more of fluorescent quantitative PCR, methylation-specific PCR, digital PCR, DNA methylation chip, targeted DNA methylation sequencing, whole genome methylation sequencing, and DNA methylation mass spectrometry.

20. A system for diagnosing esophageal cancer, wherein: The system includes a detection device, a calculation device and an output device; The detection device comprises an injector and a detector, wherein the injector is used to collect a sample to be tested from a subject, and the detector is used to detect the methylation level of the methylation biomarker according to any one of claims 1 to 16 in the sample to be tested; The computing device includes a memory and a processor, wherein the memory stores a computer program, and the processor is configured to execute the computer program stored in the memory to achieve the following determination: If the methylation level of the methylation biomarker according to any one of claims 1 to 16 in the sample is different from the methylation level of the methylation biomarker measured in a sample from a subject not suffering from esophageal cancer, it is determined that the subject corresponding to the sample has esophageal cancer; Optionally, the esophageal cancer is esophageal cancer present in a subject; optionally, the subject is a mammal; preferably, the mammal is a human; Optionally, the esophageal cancer is selected from stage I, stage II, stage III or stage IV esophageal cancer; Optionally, the subject not suffering from esophageal cancer includes a healthy subject or a subject suffering from a benign disease of the digestive system; Optionally, the sample to be tested is selected from one or more of tissue, whole blood, plasma, saliva, serum, urine, urine exfoliated cells, urine sediment, and urine supernatant.

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