Marker for detecting esophageal cancer and detection method

Abstract

Provided are a marker for detecting esophageal cancer and a detection method. Specifically, provided is the use of a reagent for evaluating the methylation status or level of a DNA region in a biological sample from an individual in the preparation of a diagnostic reagent or a diagnostic kit for diagnosing esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in an individual. The DNA region is selected from any one or more of BCAN gene or genomic BCAN sequence, LMX1B gene or genomic LMX1B sequence, a sequence at positions 89952513-89952639 of chromosome 15 of the genome, RP11-17A4.2 gene or genomic RP11-17A4.2 sequence, SPAG6 gene or genomic SPAG6 sequence, CCDC181 gene or genomic CCDC181 sequence, RNF220 gene or genomic RNF220 sequence, NKX2-5 gene or genomic NKX2-5 sequence, and SP9 gene or genomic SP9 sequence.

Description

Markers and detection methods for detecting esophageal cancer Technical Field

[0001] The present invention relates to the field of tumor gene detection, and in particular to a marker and a detection method for detecting esophageal cancer. Background Art

[0002] According to global cancer statistics in 2020, esophageal cancer ranks seventh in incidence and sixth in mortality worldwide, accounting for an average of 1 in 19 cancer deaths. The incidence rate is 2-3 times higher in men than in women, with approximately 70% of esophageal cancer cases occurring in men. There are two common subtypes of esophageal cancer: esophageal squamous cell carcinoma (ESC) and esophageal adenocarcinoma (Adenocarcinoma), which differ significantly from each other and have completely different causes. Esophageal squamous cell carcinoma (ESC) accounts for over 90% of cases in many countries, including developing countries, with the main risk factors being smoking, alcohol consumption, betel nut consumption, and the consumption of pickled vegetables. In contrast, adenocarcinoma (Adenocarcinoma) accounts for approximately two-thirds of esophageal cancer cases in high-income countries, with major risk factors being obesity, gastroesophageal reflux, and Barrett's esophagus.

[0003] Current technologies used for esophageal cancer diagnosis have very limited application in the detection and screening of early esophageal cancer, including: 1) imaging detection technologies (esophagoscopy, CT, MRI, etc.), which have high equipment costs, strong operating skills requirements, and high detection costs; 2) endoscopic biopsy of esophageal cancer, which has limited sample collection, is highly invasive, and is difficult to perform repeatedly in clinical practice, making it unsuitable for early cancer screening; 3) non-invasive testing using commonly used gastrointestinal tumor serum markers (such as CEA, CA125, CA199, and AFP, etc.), but its sensitivity is very low and cannot meet clinical detection needs.

[0004] Therefore, developing new, sensitive, and specific biomarkers and detection technologies for esophageal cancer is crucial for increasing early detection, improving treatment outcomes, and reducing mortality. Years of research have demonstrated that epigenetics plays a crucial role in the development and progression of cancer. DNA methylation / demethylation, a key mechanism of epigenetics, has been extensively studied in various tumor types, including colorectal, gastric, and lung cancers. Numerous studies have shown that the regulation of gene methylation is linked to biological mechanisms such as chromatin structure and gene expression. Changes in cellular gene methylation / demethylation occur early in tumor formation and persist throughout the development and progression of cancer, potentially providing new opportunities for cancer diagnosis and treatment. Methylation of tumor suppressor genes and demethylation of oncogenes are key molecular mechanisms in the transformation of precancerous lesions into malignant tumor cells. The underlying principle is that upon death, tumor cells release free DNA into the bloodstream. By detecting tumor-related information in the blood, such as DNA mutations, DNA methylation, miRNAs, and histone modifications, very early tumor signals can be detected. Studies have found that changes in DNA methylation may occur earlier than DNA mutations and is a more effective detection marker for early tumor screening.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a marker and a detection method for detecting esophageal cancer in order to address the deficiencies of the existing technology.

[0007] The first aspect of the present invention provides the use of primer molecules and / or probe molecules in the preparation of a detection reagent or diagnostic kit for diagnosing individual esophageal cancer, the risk of esophageal cancer, or the progression of esophageal cancer, as well as the use of a device in the preparation of a diagnostic kit for diagnosing individual esophageal cancer, the risk of esophageal cancer, or the progression of esophageal cancer; the primer molecules are identical to, complementary to, or hybridize under stringent conditions to one or more target markers and contain at least 9 consecutive nucleotides; the probe molecules hybridize under stringent conditions to the amplification products of one or more target markers; the device is used to determine the methylation state or level of at least one CpG dinucleotide of one or more target markers; wherein, the one or Multiple target markers are selected from: any one or more of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome.

[0008] In one or more embodiments, the target marker comprises the SPAG6 gene or the SPAG6 sequence of the genome and the NKX2-5 gene or the NKX2-5 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0009] In one or more embodiments, the target marker comprises the SPAG6 gene or the SPAG6 sequence of the genome and the CCDC181 gene or the CCDC181 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0010] In one or more embodiments, the target marker comprises the LMX1B gene or the LMX1B sequence of the genome and the SPAG6 gene or the SPAG6 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0011] In one or more embodiments, the target marker comprises the sequence of position 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, and the CCDC181 gene or the CCDC181 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0012] In one or more embodiments, the target marker comprises the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, and the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0013] In one or more embodiments, the target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, and the CCDC181 gene or the CCDC181 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0014] In one or more embodiments, the target marker comprises the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, and the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0015] In one or more embodiments, the target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, and the CCDC181 gene or the CCDC181 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0016] In one or more embodiments, the target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, and the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0017] In one or more embodiments, the target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, and the RNF220 gene or the RNF220 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0018] In one or more embodiments, the target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, and the NKX2-5 gene or the NKX2-5 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0019] In one or more embodiments, the target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, and the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0020] In one or more embodiments, the target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, and the RNF220 gene or the RNF220 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0021] In one or more embodiments, the target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, and the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0022] In one or more embodiments, the target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, and the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0023] In one or more embodiments, the target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0024] In one or more embodiments, the target marker comprises the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0025] In one or more embodiments, the target marker comprises the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0026] In one or more embodiments, the target marker comprises the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0027] In one or more embodiments, the target marker comprises the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises the RNF220 gene or the RNF220 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0028] In one or more embodiments, the target marker comprises the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0029] In one or more embodiments, the target marker comprises the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

[0030] In one or more embodiments, the Hg19 coordinates of each gene or intergenic region are as follows: BCAN gene, chr1: 156611182-156629324; LMX1B gene, chr9: 129376722-129463311; 15: 89952513-89952639, chr15: 89952513-89952639; RP11-17A4.2 gene, chr8: 57358366-5 7464626; SPAG6 gene, chr10: 22634399-22743153; CCDC181 gene, chr1: 169364108-169429907; RNF220 gene, chr1: 44870866-45117396; NKX2-5 gene, chr5: 172659112-172662360; SP9 gene, chr2: 175199674-175203220.

[0031] In one or more embodiments, the one or more target markers are one or more target regions obtained by amplifying each gene or intergenic region using the following primers:

[0032] Target region of the BCAN gene: a fragment of the BCAN gene amplified using primers representing sequences shown in SEQ ID NOs: 1 and 2, or sequences having at least 90% identity to SEQ ID NOs: 1 and 2;

[0033] Target region of the LMX1B gene: a fragment of the LMX1B gene amplified using the sequences shown in SEQ ID NOs: 3 and 4, or sequences having at least 90% identity to SEQ ID NOs: 3 and 4 as primers;

[0034] Target region 15:89952513-89952639: a fragment amplified using primers representing sequences shown in SEQ ID NOs:5 and 6 or sequences having at least 90% identity to SEQ ID NOs:5 and 6;

[0035] Target region of the RP11-17A4.2 gene: a fragment of the RP11-17A4.2 gene amplified using primers having sequences represented by SEQ ID NOs: 7 and 8 or sequences having at least 90% identity thereto;

[0036] Target region of the SPAG6 gene: a fragment of the SPAG6 gene amplified using primers representing sequences shown in SEQ ID NOs: 9 and 10, or sequences having at least 90% identity to SEQ ID NOs: 9 and 10;

[0037] Target region of the CCDC181 gene: a fragment of the CCDC181 gene amplified using primers representing sequences shown in SEQ ID NOs: 11 and 12, or sequences having at least 90% identity thereto;

[0038] Target region of the RNF220 gene: a fragment of the RNF220 gene amplified using primers representing sequences shown in SEQ ID NOs: 13 and 14, or sequences having at least 90% identity thereto;

[0039] Target region of the NKX2-5 gene: a fragment of the NKX2-5 gene amplified using primers using the sequences shown in SEQ ID NOs: 15 and 16 or sequences having at least 90% identity to SEQ ID NOs: 15 and 16;

[0040] Target region of the SP9 gene: a fragment of the SP9 gene amplified using the sequences shown in SEQ ID NOs: 17 and 18 or sequences having at least 90% identity with SEQ ID NOs: 17 and 18 as primers.

[0041] In one or more embodiments, the Hg19 coordinates of the target region of each gene or intergenic region are as follows: the target region of the BCAN gene, chr1: 156611866-156611966; the target region of the LMX1B gene, chr9: 129377872-129377947; the target region of the RP11-17A4.2 gene, chr8: 57358572-57358573; 7358671; target region of SPAG6 gene, chr10: 22634425-22634494; target region of CCDC181 gene, chr1: 169396617-169396725; target region of RNF220 gene, chr1: 44883356-44883454; target region of NKX2-5 gene, chr5: 172659753-172659839; target region of SP9 gene, chr2: 175202025-175202158.

[0042] In one or more embodiments, the primer molecules for each gene or intergenic region are: BCAN: SEQ ID NOs: 1 and 2, or primer molecules that hybridize under stringent conditions with the fragments amplified therefrom; LMX1B: SEQ ID NOs: 3 and 4, or primer molecules that hybridize under stringent conditions with the fragments amplified therefrom; 15:89952513-89952639: SEQ ID NOs: 5 and 6, or primer molecules that hybridize under stringent conditions with the fragments amplified therefrom; RP11-17A4.2: SEQ ID NOs: 7 and 8, or primer molecules that hybridize under stringent conditions with the fragments amplified therefrom; SPAG6: SEQ ID NOs: 9 and 10, or primer molecules that hybridize under stringent conditions with the fragments amplified therefrom; CCDC181: SEQ ID NOs: 11 and 12, or primer molecules that hybridize under stringent conditions with the fragments amplified therefrom; RNF220: SEQ ID NOs: 13 and 14, or primer molecules that hybridize under stringent conditions with the fragments amplified therefrom; NKX2-5: SEQ ID NOs: NO: 15 and 16, or primer molecules that hybridize to the fragments amplified therefrom under stringent conditions; SP9: SEQ ID NO: 17 and 18, or primer molecules that hybridize to the fragments amplified therefrom under stringent conditions.

[0043] In one or more embodiments, the probe molecules for the amplified products of each gene or intergenic region are: BCAN: SEQ ID NO: 21 or a sequence having at least 90% identity thereof; LMX1B: SEQ ID NO: 22 or a sequence having at least 90% identity thereof; 15:89952513-89952639: SEQ ID NO: 23 or a sequence having at least 90% identity thereof; RP11-17A4.2: SEQ ID NO: 24 or a sequence having at least 90% identity thereof; SPAG6: SEQ ID NO: 25 or a sequence having at least 90% identity thereof; CCDC181: SEQ ID NO: 26 or a sequence having at least 90% identity thereof; RNF220: SEQ ID NO: 27 or a sequence having at least 90% identity thereof; NKX2-5: SEQ ID NO: 28 or a sequence having at least 90% identity thereof; SP9: SEQ ID NO: 29 or a sequence having at least 90% identity thereof.

[0044] In one or more embodiments, the detection reagent or diagnostic kit further comprises primer molecules and / or probe molecules for detecting the internal reference gene ACTB; preferably, the primer molecules for the internal reference gene ACTB are sequences shown in SEQ ID NOs: 19 and 20 or sequences that hybridize under stringent conditions to a fragment of the ACTB gene amplified using SEQ ID NOs: 19 and 20 as primers, and the probe for the internal reference gene ACTB comprises the sequence shown in SEQ ID NO: 30 or a sequence that is at least 90% identical thereto.

[0045] The present invention also provides a diagnostic reagent or diagnostic kit for detecting the methylation state or methylation level of at least one CpG dinucleotide of a target marker to diagnose esophageal cancer, the risk of esophageal cancer or the progression of esophageal cancer, which comprises a primer molecule and / or a probe molecule; wherein the primer molecule is identical to, complementary to or hybridizes under stringent conditions to one or more target markers and comprises at least 9 consecutive nucleotides, and the probe molecule hybridizes under stringent conditions to the amplification product of one or more target markers, wherein the one or more target markers are selected from: BCAN gene or BCAN sequence of genome , LMX1B gene or genomic LMX1B sequence, the sequence of chromosome 15 of the genome at position 89952513-89952639, RP11-17A4.2 gene or genomic RP11-17A4.2 sequence, SPAG6 gene or genomic SPAG6 sequence, CCDC181 gene or genomic CCDC181 sequence, RNF220 gene or genomic RNF220 sequence, NKX2-5 gene or genomic NKX2-5 sequence, SP9 gene or genomic SP9 sequence, any one or more of them.

[0046] In one or more embodiments, the one or more target markers are as described in any embodiment of the first aspect of this invention; preferably, the primer molecules and probe molecules are as described in any embodiment of the first aspect of this invention.

[0047] In one or more embodiments, the diagnostic reagent or diagnostic kit further comprises primer molecules and / or probe molecules for detecting the internal reference gene ACTB; preferably, the primer molecules for the internal reference gene ACTB are sequences shown in SEQ ID NOs: 19 and 20 or sequences that hybridize under stringent conditions to a fragment of the ACTB gene amplified using SEQ ID NOs: 19 and 20 as primers, and the probe for the internal reference gene ACTB comprises the sequence shown in SEQ ID NO: 30 or a sequence that is at least 90% identical thereto.

[0048] In one or more embodiments, the diagnostic kit further comprises one or more substances selected from the group consisting of: PCR buffer, polymerase, dNTP, restriction endonuclease, enzyme digestion buffer, fluorescent dye, fluorescence quencher, fluorescence reporter, exonuclease, alkaline phosphatase, internal standard, control, KCl, MgCl2 and (NH4)2SO4.

[0049] In one or more embodiments, reagents for detecting DNA methylation also include reagents used in one or more of the following methods: PCR based on bisulfite conversion, DNA sequencing, methylation-sensitive restriction endonuclease analysis, fluorescence quantification, methylation-sensitive high-resolution melting curve method, chip-based methylation profiling and mass spectrometry.

[0050] In one or more embodiments, the reagent is selected from one or more of the following: bisulfite and its derivatives, fluorescent dyes, fluorescence quenchers, fluorescent reporters, internal standards, and controls.

[0051] The present invention also provides the use of at least one reagent or a panel of reagents that distinguishes between methylated and unmethylated CpG dinucleotides in at least one target region of genomic DNA in the preparation of a kit for the detection and / or classification of esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in an individual, wherein the method comprises contacting genomic DNA isolated from a biological sample of the individual with the at least one reagent or panel of reagents, wherein the target region is identical or complementary to a sequence of at least 16 consecutive nucleotides of one or more markers of interest, wherein the consecutive nucleotides comprise at least one CpG dinucleotide sequence, thereby at least partially providing for the detection and / or classification of esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer. Category, wherein the one or more target markers are selected from: any one or more of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of chromosome 15 of the genome at positions 89952513-89952639, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome.

[0052] In one or more embodiments, the one or more target markers are as described in any embodiment of the first aspect herein.

[0053] The present invention also provides the use of one or more reagents that convert unmethylated cytosine base at position 5 to uracil or other bases detectably different from cytosine in hybridization properties, an amplification enzyme, and at least one primer comprising at least 9 consecutive nucleotides in the preparation of a kit for a method for detecting and / or classifying esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in an individual, wherein the method comprises:

[0054] a) isolating genomic DNA from a biological sample of the individual;

[0055] b) treating the genomic DNA or fragments thereof of a) with the one or more reagents;

[0056] c) contacting the treated genomic DNA or treated fragments thereof with the amplification enzyme and the at least one primer that is identical to, complementary to, or hybridizes under stringent conditions to one or more target markers, wherein the treated genomic DNA or fragments thereof is amplified to produce at least one amplification product or is not amplified; and

[0057] d) determining the methylation status or level of at least one CpG dinucleotide of the one or more target markers, or a mean or value reflecting the average methylation status or level of multiple CpG dinucleotides of the one or more target markers, based on the presence or nature of the amplicon, thereby at least in part detecting and / or classifying esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in the individual;

[0058] Wherein, the one or more target markers are selected from: BCAN gene or BCAN sequence of the genome, LMX1B gene or LMX1B sequence of the genome, sequence of chromosome 15 of the genome at positions 89952513-89952639, RP11-17A4.2 gene or RP11-17A4.2 sequence of the genome, SPAG6 gene or SPAG6 sequence of the genome, CCDC181 gene or CCDC181 sequence of the genome, RNF220 gene or RNF220 sequence of the genome, NKX2-5 gene or NKX2-5 sequence of the genome, SP9 gene or SP9 sequence of the genome, any one or more of them.

[0059] In one or more embodiments, the one or more target markers are as described in any embodiment of the first aspect herein; preferably, the primer molecules are as described in any embodiment of the first aspect herein.

[0060] In one or more embodiments, in step b), the genomic DNA or fragments thereof are treated with a reagent selected from bisulfite, acid sulfite, metabisulfite and combinations thereof and an enzyme that can distinguish between methylated and unmethylated cytosine.

[0061] In one or more embodiments, in c), the contacting or amplification of nucleic acid molecules is performed by using a thermostable DNA polymerase as the amplification enzyme, using a polymerase lacking 5'-3' exonuclease activity, using polymerase chain reaction, and / or generating an amplification product with a detectable label.

[0062] In one or more embodiments, the contacting or amplifying in c) comprises using methylation-specific primers.

[0063] The present invention also provides use of one or more methylation-sensitive restriction enzymes and amplification enzymes and at least one primer comprising at least 9 consecutive nucleotides in preparing a kit for detecting and / or classifying esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in an individual, wherein the primers are identical to, complementary to, or hybridize under stringent conditions to one or more target markers; the method comprising:

[0064] a) isolating genomic DNA from a biological sample of the individual;

[0065] b) digesting the genomic DNA or fragment thereof described in a) with the one or more methylation-sensitive restriction enzymes, and contacting the resulting digestion product with the amplification enzyme and the at least one primer; and

[0066] c) determining the methylation status or level of at least one CpG dinucleotide of the one or more target markers based on the presence or nature of the amplification product, thereby at least in part detecting and / or classifying esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in the individual;

[0067] Wherein, the one or more target markers are selected from: BCAN gene or BCAN sequence of the genome, LMX1B gene or LMX1B sequence of the genome, sequence of chromosome 15 of the genome at positions 89952513-89952639, RP11-17A4.2 gene or RP11-17A4.2 sequence of the genome, SPAG6 gene or SPAG6 sequence of the genome, CCDC181 gene or CCDC181 sequence of the genome, RNF220 gene or RNF220 sequence of the genome, NKX2-5 gene or NKX2-5 sequence of the genome, SP9 gene or SP9 sequence of the genome, any one or more of them.

[0068] In one or more embodiments, the one or more target markers are as described in any embodiment of the first aspect herein; preferably, the primer molecules are as described in any embodiment of the first aspect herein.

[0069] In one or more embodiments, the presence or absence of an amplification product is determined by hybridizing at least one nucleic acid or peptide nucleic acid that is identical or complementary to a fragment of at least 16 bases long selected from the sequence of the one or more target markers.

[0070] The present invention also provides the use of a treated nucleic acid derived from one or more target markers in the preparation of a kit for diagnosing esophageal cancer, the risk of developing esophageal cancer or the progression of esophageal cancer, wherein the treatment is suitable for converting at least one unmethylated cytosine base of the one or more target markers to uracil or other bases that are detectably different from cytosine in hybridization, and the one or more target markers are selected from: any one or more of the BCAN gene or genomic BCAN sequence, the LMX1B gene or genomic LMX1B sequence, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome.

[0071] In one or more embodiments, the one or more target markers are as described in any one of the embodiments of the first aspect herein.

[0072] The present invention also provides an apparatus for detecting and diagnosing esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in an individual, the apparatus comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein when the processor executes the program, the following steps are implemented: (1) obtaining the methylation level or methylation status of at least one CpG dinucleotide of one or more target markers in a sample, and (2) judging esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer based on the methylation level or methylation status of (1);

[0073] Wherein, the one or more target markers are selected from: BCAN gene or BCAN sequence of the genome, LMX1B gene or LMX1B sequence of the genome, sequence of chromosome 15 of the genome at positions 89952513-89952639, RP11-17A4.2 gene or RP11-17A4.2 sequence of the genome, SPAG6 gene or SPAG6 sequence of the genome, CCDC181 gene or CCDC181 sequence of the genome, RNF220 gene or RNF220 sequence of the genome, NKX2-5 gene or NKX2-5 sequence of the genome, SP9 gene or SP9 sequence of the genome, any one or more of them.

[0074] In one or more embodiments, the one or more target markers are as described in any embodiment of the first aspect herein.

[0075] The present invention also provides the use of a reagent for assessing the methylation state or level of a DNA region in a biological sample from an individual in the preparation of a diagnostic reagent or diagnostic kit for diagnosing esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in an individual; wherein the DNA region is selected from: any one or more of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1: ROC curves of 9 single methylation sites in the training set of Example 2.

[0077] FIG2 : ROC curves of 9 single methylation sites in the validation set of Example 3.

[0078] Figure 3: AUC of combined sites in the training set of Example 2.

[0079] Figure 4: AUC of the combined sites in the validation set of Example 3. DETAILED DESCRIPTION

[0080] Although the present application discloses various aspects and various embodiments of the present application, those skilled in the art can make various equivalent changes or modifications without departing from the spirit and scope of the present application. The various aspects and various embodiments disclosed in the present application are exemplary and are not intended to limit the scope of the present application. The actual scope of protection of the present application is subject to the claims. Unless otherwise indicated, all technical and scientific terms used in this application are the meanings commonly understood by those skilled in the art. All references, patents and patent applications cited in this application are incorporated into this application by reference.

[0081] It should be noted that, in the specification and claims of this application, the singular forms "a", "an", and "the" include plural forms thereof unless the context indicates otherwise. Thus, for example, "an agent" includes a plurality of agents.

[0082] In the specification and claims of this application, unless otherwise stated, the terms "comprise", "include" or "contain" mean that the listed values, steps or components are included, but other values, steps or components are not excluded.

[0083] After in-depth research, the inventors discovered some target markers related to esophageal cancer, which include: BCAN gene or BCAN sequence of the genome, LMX1B gene or LMX1B sequence of the genome, sequence of chromosome 15 at positions 89952513-89952639 (or 15:89952513-89952639), RP11-17A4.2 (also known as PENK) gene or RP11-17A4.2 (PENK) sequence of the genome, SPAG6 gene or SPAG6 sequence of the genome, CCDC181 gene or CCDC181 sequence of the genome, RNF220 gene or RNF220 sequence of the genome, NKX2-5 gene or NKX2-5 sequence of the genome, SP9 gene or SP9 sequence of the genome. By detecting the methylation level of one or more target markers in a biological sample containing DNA from an individual, the presence of an esophageal tumor can be confirmed, the risk of esophageal tumor formation can be assessed, and / or the progression of an esophageal tumor can be assessed.

[0084] I. Target markers and their target areas

[0085] As used herein, the term "target marker" refers to such a nucleic acid or gene region of interest: its methylation level indicates the presence of an esophageal tumor, the risk of formation of an esophageal tumor, and / or the progression of an esophageal tumor. The term "target marker" should be considered to include all transcript variants thereof and all promoters and regulatory elements thereof. As understood by those skilled in the art, it is known that certain genes exhibit allelic variation or single nucleotide polymorphism ("SNP") between individuals. SNP includes insertions and deletions of simple repetitive sequences (such as dinucleotides and trinucleotide repeats) of varying lengths. Therefore, the application should be understood to extend to all forms of markers / genes produced by any other mutation, polymorphism, or allelic variation. In addition, it should be understood that the term "target marker" should include both the positive strand sequence of the marker or gene and the antisense strand sequence of the marker or gene.

[0086] In addition, the term "target marker" is also interpreted to include both 1) the original marker found in a biological sample or genomic DNA (in a specific methylation state), and 2) its processed sequence (e.g., the corresponding region after bisulfite conversion). The corresponding region after bisulfite conversion differs from the target marker in the genomic sequence in that one or more unmethylated cytosine residues are converted to uracil, thymine, or other bases that have different hybridization behavior from cytosine.

[0087] It should be understood herein that the target markers BCAN, LMX1B, sequence 89952513-89952639 of chromosome 15 (also referred to herein as 15:89952513-89952639 or T3 target), RP11-17A4.2 (also referred to as PENK), SPAG6, CCDC181, RNF220, NKX2-5, and SP9 involved in the various products, uses, and methods described herein can be described by reference to both their names and their chromosomal coordinates. The chromosomal coordinates are consistent with the Hg19 version of the human genome database released in February 2009 (referred to herein as "Hg19 coordinates"). It should be understood that the sequences of a gene and its genome described herein also include fragments of each gene containing at least one CpG dinucleotide sequence. In some embodiments, the fragment is the target region of each gene described herein.

[0088] In some embodiments, the target regions of each gene are:

[0089] (1) a fragment of the BCAN gene amplified using primers derived from SEQ ID NOs: 1 and 2 or sequences having at least 90% identity thereto;

[0090] (2) a fragment of the LMX1B gene amplified using primers derived from SEQ ID NOs: 3 and 4 or sequences having at least 90% identity thereto;

[0091] (3) a fragment of sequence 15:89952513-89952639 obtained by amplifying using primers of SEQ ID NO:5 and 6 or sequences having at least 90% identity thereto;

[0092] (4) a fragment of the RP11-17A4.2 gene amplified using primers derived from SEQ ID NOs: 7 and 8 or sequences having at least 90% identity thereto;

[0093] (5) a fragment of the SPAG6 gene amplified using primers derived from SEQ ID NOs: 9 and 10 or sequences having at least 90% identity thereto;

[0094] (6) a fragment of the CCDC181 gene amplified using primers derived from SEQ ID NOs: 11 and 12 or sequences having at least 90% identity thereto;

[0095] (7) a fragment of the RNF220 gene amplified using primers derived from SEQ ID NOs: 13 and 14 or sequences having at least 90% identity thereto;

[0096] (8) a fragment of the NKX2-5 gene amplified using primers derived from SEQ ID NOs: 15 and 16 or sequences having at least 90% identity thereto; and

[0097] (9) a fragment of the SP9 gene amplified using primers derived from SEQ ID NOs: 17 and 18 or sequences having at least 90% identity thereto;

[0098] In a preferred embodiment, the Hg19 coordinates of the gene and its target region are defined as follows:

[0099] The target marker of the present invention also includes the upstream 5 kb of each starting site of each region and / or the downstream 5 kb of each end site, preferably includes the upstream 1 kb, 500 bp, 200 bp, 100 bp or 50 bp or less of each starting site of each region and / or the downstream 5 kb, 1 kb, 500 bp, 200 bp, 100 bp or 50 bp or less of each end site. The specific nucleotide sequence of the Hg19 coordinates, as well as the upstream 5 kb of each starting site of each region and the downstream 5 kb of each end site, can be obtained in public databases (e.g., UCSC Genome Browser, Ensemble, and NCBI websites).

[0100] The target markers of the present invention (such as a gene and its genomic sequence, or a fragment of each gene containing at least one CpG dinucleotide sequence, or the target region, or a sequence containing a gene intergenic region) also include corresponding regions obtained after non-enzymatic conversion (such as the corresponding region after bisulfite conversion) and enzymatic conversion (such as MSRE conversion).

[0101] In some embodiments, the target markers of the present invention also include various variants of each of the above-mentioned genes or target regions. Variants include nucleic acid sequences from the same region that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity (i.e., having one or more deletions, insertions, substitutions, reverse sequences, etc.) to the genes or regions described herein. Therefore, the present application should be understood to extend to such variants that achieve the same results, despite the fact that the actual nucleic acid sequences between individuals have slight genetic variations. In some embodiments, this column of variants is a naturally occurring variant.

[0102] As used herein, the term "percentage (%) of sequence identity" refers to the percentage of identical amino acid (or nucleic acid) residues in a candidate sequence and the amino acid (or nucleic acid) residues in a reference sequence after sequence alignment, wherein intervals may be introduced (if necessary) to maximize the number of identical amino acids (or nucleic acids). In other words, the percentage (%) of sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) in the candidate sequence or the reference sequence by the total number of amino acid residues (or bases), whichever is shorter, in the amino acid sequence. Conservative substitutions of amino acid residues may or may not be considered to be identical residues. The percentage of amino acid (or nucleic acid) sequence identity can be determined by, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), see also Altschul SF et al. J. Mol. Biol. 215: 403-410 (1990); Stephen F et al. Nucleic Acids Res. 25: 3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute), see also Higgins DG et al. Methods in Enzymology 266: 383-402 (1996); Larkin MA et al. Bioinformatics (Oxford England) 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the tool, or can customize parameters suitable for the alignment (e.g., by selecting an appropriate algorithm).

[0103] II. Source and Preparation of Target Markers

[0104] Herein, the target marker can be from a biological sample of any individual of interest. The term "individual" as used herein includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. "Individual" can also be livestock, such as cattle, pigs, sheep, poultry and horses; or rodents, such as rats, mice; or non-human primates, such as apes, monkeys, rhesus macaques; or domesticated animals, such as dogs or cats. In some embodiments, the individual is a human or a non-human primate. In some embodiments, the individual is a human. In this application, "individual," "object," and "subject" are used interchangeably.

[0105] It should be understood that the sequences given in Section 1 above are human sequences. When sequences of non-human animals are involved, the corresponding positions and sequences of the genes in the non-human animal genomes can be easily determined using existing technologies.

[0106] The term "biological sample" as used herein refers to a biological composition obtained or derived from an individual, comprising cells and / or other molecular entities (e.g., DNA) to be characterized or identified based on physical, biochemical, chemical, and / or physiological characteristics. Biological samples include, but are not limited to, cells, tissues, organs, and / or biological fluids of an individual obtained by any method known to those skilled in the art. In some embodiments, the biological sample is selected from the group consisting of: histological sections, tissue biopsies, paraffin-embedded tissues, body fluids, surgical resection specimens, isolated blood cells, cells isolated from blood, and any combination thereof. In one or more embodiments, the tissue is esophageal tissue; in one or more embodiments, the cells are esophageal cells. In one or more embodiments, the cells are buffy coat cells. In some embodiments, the body fluid is selected from the group consisting of: whole blood, serum, plasma, and any combination thereof. The selection of the most appropriate sample will depend on the nature of the context. In some embodiments, the biological sample is whole blood from an individual. In some embodiments, the biological sample is plasma from an individual. Those skilled in the art are aware of various methods for preparing plasma from whole blood. For example, in some embodiments, plasma is obtained by centrifuging whole blood from an individual one, two, three, four, five, or more times.

[0107] DNA to be detected can be separated from the biological sample. DNA to be detected can be separated and purified from a biological sample using the whole bag of tricks known in the art. Commercially available kits can be used to separate and purify. For example, DNA can be isolated from cells and tissues in the following manner: the nucleic acid components obtained by pyrolysis of raw materials, partial use of protein degrading enzymes, purification under high denaturation and reducing conditions by phenol / chloroform extraction process, and nucleic acid recovery from aqueous phase by dialysis or ethanol precipitation (see, for example, Sambrook J. Fritsch EFin T. Maniatis CSH Molecular Cloning 1989). For another example, there are many reagent systems that are particularly suitable for purifying DNA fragments from agarose gels, separating plasmid DNA from bacterial lysates, and separating longer-chain nucleic acids (genomic DNA, total cell RNA) from blood, tissue or cell culture.

[0108] In some embodiments, the DNA extraction reagent used consists of a lysis buffer, a resuspension buffer, a cleaning buffer, and an elution buffer. The lysis buffer consists of a protein denaturant, a detergent, a pH buffer, and a nuclease inhibitor. The resuspension buffer consists of a protein denaturant and a pH buffer. The cleaning buffer consists of a protein denaturant, a nuclease inhibitor, a detergent, a pH buffer, and ethanol. The elution buffer consists of a nuclease inhibitor and a pH buffer. An exemplary protein denaturant is guanidine hydrochloride; an exemplary detergent is Tween 20; an exemplary pH buffer is Tris-HCl; and an exemplary nuclease inhibitor is EDTA.

[0109] In some embodiments, the DNA contained in the biological sample includes genomic DNA. The term "genomic DNA" as used herein refers to DNA comprising the complete genome of a cell or organism and fragments or portions thereof. Genomic DNA is a large segment of DNA (e.g., longer than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 300 kb) derived from an individual and may have natural modifications, such as DNA methylation.

[0110] In some embodiments, the DNA contained in the biological sample includes cellular DNA. As used herein, the term "cellular DNA" refers to DNA present in cells, or DNA obtained from cells in vivo and isolated in vitro, or otherwise manipulated in vitro, as long as the DNA is not removed from cells in vivo.

[0111] In some embodiments, the DNA contained in the biological sample includes extracellular free DNA. The term "extracellular free DNA" used herein refers to DNA fragments present outside the cells in the body. The term can also be used to refer to DNA fragments obtained from extracellular sources in the body and separated or manipulated in vitro. The DNA fragments in the extracellular free DNA generally have a length of about 50 to 200 bp, such as a length of 50 to 150 bp. In some embodiments, the extracellular free DNA may be substantially free of individual cellular DNA. For example, the extracellular free DNA may comprise less than about 1000 ng / mL, less than about 100 ng / mL, less than about 10 ng / mL, or less than about 1 ng / mL of cellular DNA.

[0112] Extracellular free DNA can be prepared by using conventional techniques known in the art. For example, the extracellular free DNA of a blood sample can be obtained by centrifuging a blood sample at a speed of about 200-20000g, about 200-10000g, about 200-5000g, about 300-4000g, etc. for about 3-30 minutes, about 3-15 minutes, about 3-10 minutes, about 3-5 minutes. For example, in some embodiments, the extracellular free DNA of a blood sample can be obtained by centrifuging the plasma or serum of an individual one, two, three, four, five times or more. In some embodiments, in order to separate cells and fragments thereof from the cell-free component comprising soluble DNA, the biological sample can be obtained by microfiltration. Generally speaking, microfiltration can be carried out by using a filter, for example, a membrane filter of 0.1 micron to 0.45 micron, such as a membrane filter of 0.22 micron.

[0113] In some embodiments, commercially available DNA extraction products are used to extract extracellular DNA from whole blood, serum, or plasma for analysis. This extraction method is reported to provide high recovery rates (>50%) of circulating DNA, and some products (e.g., the QIAamp Circulating Nucleic Acid Kit from Qiagen) are reported to be able to extract small DNA fragments. Typical sample volumes used are 1-5 mL of serum or plasma.

[0114] III. Base conversion

[0115] Herein, DNA methylation is a biological process in which a methyl group is added to a DNA molecule (e.g., to one or more cytosine bases of a DNA molecule) (e.g., by the action of a DNA methyltransferase). In mammals, DNA methylation occurs at the 5' position of a cytosine-phosphate-guanine (CpG) dinucleotide (i.e., a "CpG site") and, when present in a 5'-CpG-3' dinucleotide in the promoter or first exon of a gene, results in epigenetic inactivation of the gene. It has been well documented that DNA methylation plays an important role in regulating gene expression, tumorigenesis, and other genetic and epigenetic diseases.

[0116] As used herein, the term "methylated cytosine residue" refers to a derivative of a cytosine residue in which one methyl group is attached to a carbon atom of the cytosine ring (e.g., C5). The term "unmethylated cytosine residue" refers to an underivatized cytosine residue in which, in contrast to a "methylated cytosine residue," no methyl group is attached to a carbon atom of the cytosine ring (e.g., C5). A CpG site in which the cytosine residue is methylated is a methylated CpG site, while a CpG site in which the cytosine residue is not methylated is an unmethylated CpG site.

[0117] As described herein, conversion can occur between bases in DNA or RNA. "Conversion," "cytosine conversion," or "CT conversion" as described herein refers to the process of treating DNA using non-enzymatic or enzymatic methods to convert unmodified cytosine bases (cytosine, C) into bases that do not bind to guanine (G), such as uracil bases (uracil, U). Some reagents are capable of distinguishing between unmethylated and methylated CpG sites in DNA, thereby obtaining treated DNA. The reagent can selectively act on unmethylated cytosine residues but not significantly act on methylated cytosine residues. Alternatively, the reagent can selectively act on methylated cytosine residues but not significantly act on unmethylated cytosine residues. For example, some reagents can selectively convert unmethylated cytosine residues into uracil, thymine, or another base that is different from cytosine in hybridization, while methylated cytosine residues remain in an unconverted state; for another example, some reagents can selectively cleave methylated residues or selectively cleave unmethylated residues. Thereby, the original DNA is converted into the treated DNA in a manner depending on whether it is methylated or not, so that the treated DNA can be distinguished from the original DNA by its hybridization behavior.

[0118] As used herein, "treated DNA," "treated sequence," and "treated fragment" refer to DNA, nucleic acid sequences, and gene fragments that have been treated with a reagent that can distinguish between unmethylated and methylated CpG sites in DNA, nucleic acid sequences, and gene fragments.

[0119] More specifically, cytosine conversion can be performed using non-enzymatic or enzymatic methods. Exemplarily, non-enzymatic methods include bisulfite or bisulfate treatment. In some embodiments, the reagent used in the non-enzymatic method includes a bisulfite reagent. As used herein, the term "bisulfite reagent" refers to a reagent comprising bisulfite, bisulfite ions, or any combination thereof, which can be used to distinguish methylated and unmethylated CpG dinucleotide sequences, such as those disclosed herein. In this application, treating DNA with a bisulfite reagent is also described as a "bisulfite reaction" or "bisulfite treatment," which refers to a reaction that converts unmethylated cytosine residues, particularly in the presence of bisulfite ions, where unmethylated cytosine residues in nucleic acids are converted to uracil bases, thymine bases, or other bases that differ from cytosine in hybridization behavior, while methylated cytosine residues are not significantly converted. In other words, bisulfite treatment can be used to distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides. The bisulfite reaction for detecting methylated cytosine residues is described in detail in Frommer M. et al. Proc Natl Acad Sci USA 89 (1992) 1827-31 and Grigg G. Clark S. Bioessays 16 (1994) 431-6. The bisulfite reaction includes a deamination step and a desulfonation step (see Grigg and Clark supra). The statement “methylated cytosine residues are not significantly converted” does not exclude that a very small percentage (e.g., less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, less than 15%, less than 16%, less than 17%, less than 18%, less than 19%, less than 20%) of the methylated cytosine residues are converted to uracil, thymine, or other bases that differ from cytosine in hybridization behavior, although it is intended that only unmethylated cytosine residues are converted.

[0120] Those skilled in the art will know how to perform a bisulfite treatment, in particular the deamination and desulfonation steps, by reference, for example, to Frommer M. et al. (supra) or Grigg and Clark (supra), which disclose the basic parameters of a bisulfite treatment. The effects of incubation time and temperature on the efficiency of deamination, as well as parameters that influence DNA degradation, are disclosed.

[0121] In some embodiments, the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite ion, and any combination thereof. In some embodiments, the bisulfite reagent is sodium bisulfite. In some embodiments, bisulfite reagents are commercially available, such as MethylCode™ Bisulfite Conversion Kit, EpiMark™ Bisulfite Conversion Kit, EpiJET™ Bisulfite Conversion Kit, EZDNAMethylation-Gold™ Kit, and the like. In some embodiments, the bisulfite reaction is performed according to the kit's instructions.

[0122] Exemplary enzymatic methods include deaminase treatment, such as those from NEB Enzymatic Methyl-seq Conversion Module; and using a reagent to selectively cleave unmethylated residues but not cleave methylated residues, or selectively cleave methylated residues but not cleave unmethylated residues. Preferably, the reagent is a methylation-sensitive restriction enzyme (MSRE).

[0123] The term "methylation-sensitive restriction enzyme" refers to an enzyme that selectively digests nucleic acids based on the methylation state of its recognition site. For restriction enzymes that specifically shear when the recognition site is not methylated or hemimethylated, shearing will not occur, or shearing will be significantly reduced in efficiency when the recognition site is methylated. For restriction enzymes that specifically shear when the recognition site is methylated, shearing will not occur, or shearing will be significantly reduced in efficiency when the recognition site is not methylated. In some embodiments, the recognition sequence of the methylation-sensitive restriction enzyme contains a CG dinucleotide (e.g., cgcg or cccggg). In some embodiments, when the cytosine in the CG dinucleotide is methylated at the C5 carbon atom, the methylation-sensitive restriction enzyme does not shear.

[0124] Exemplary MSREs are selected from the group consisting of HpaII enzyme, SalI enzyme, enzyme, ScrFI enzyme, BbeI enzyme, NotI enzyme, SmaI enzyme, XmaI enzyme, MboI enzyme, BstBI enzyme, ClaI enzyme, MluI enzyme, NaeI enzyme, NarI enzyme, PvuI enzyme, SacII enzyme, HhaI enzyme, and any combination thereof.

[0125] Methylation is determined using methods known in the art, such as, but not limited to, differential methylation hybridization ("DMH"), using a methylation-sensitive restriction enzyme or a panel of restriction enzyme reagents comprising a methylation-sensitive restriction enzyme that can distinguish between methylated and unmethylated CpG dinucleotides in the target region.

[0126] In some embodiments, the DNA in the biological sample can be cut before being treated with a methylation-sensitive restriction enzyme. Such methods are known in the art and can include both physical and enzymatic methods. Particularly preferably, one or more restriction enzymes that are insensitive to methylation and whose recognition sites are rich in AT and do not comprise CG dinucleotides are used. This type of enzyme is used to preserve the CpG sites and CpG enrichment regions in the DNA fragmentation. In some embodiments, this type of restriction enzyme is selected from MseI enzyme, BfaI enzyme, Csp6I15 enzyme, Tru1I enzyme, Tru9I enzyme, MaeI enzyme, XspI enzyme, and any combination thereof.

[0127] The transformed DNA is optionally purified. Methods for purifying DNA suitable for use herein are well known in the art.

[0128] IV. Methylation Analysis

[0129] The methylation status or methylation level of at least one CpG dinucleotide in any one, any two, any three, any four, any five, any six, any seven, any eight, or all nine target markers described herein, or in their target regions, can be detected to determine the presence of esophageal tumors, assess the risk of developing esophageal tumors, and / or assess the progression of esophageal tumors. The detection reagents and diagnostic kits described herein can be used to detect the methylation status or methylation level.

[0130] Herein, esophageal cancer has a meaning well known in the art, including esophageal squamous cell carcinoma and esophageal adenocarcinoma.

[0131] Herein, "methylation state" refers 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. In some embodiments, if the nucleic acid is not methylated at a specific locus (e.g., a locus of a specific single CpG dinucleotide) or a specific combination of loci, the nucleic acid can be characterized as "unmethylated", even if it is methylated at other loci of the same gene or molecule. Therefore, the methylation state describes the state of methylation of a nucleic acid (e.g., a genomic sequence or a target marker as described herein).

[0132] In addition, methylation state refers to the feature that the nucleic acid segment at specific genomic locus is relevant to methylation.Such feature includes but is not limited to whether any cytosine (C) residue in this DNA sequence is methylated, the position of one or more methylated C residues, the frequency or percentage of methylated C in any specific region of running through nucleic acid and the methylation allele difference that causes due to the difference of for example allele starting point." methylation state " is included in the relative concentration, absolute concentration or pattern of methylated C or unmethylated C in any specific region of running through nucleic acid in biological sample.For example, if one or more cytosine (C) residues in nucleic acid sequence are methylated, then it can be referred to as " hypermethylation " or have " increased methylation ", and if one or more cytosine (C) residues in DNA sequence are unmethylated, then it can be referred to as " demethylation " or have " reduced methylation ".Similarly, if one or more cytosine (C) residues in nucleic acid sequence are methylated compared with another nucleic acid sequence (for example from different regions or from different individuals etc.), then this sequence is considered to be hypermethylated or have increased methylation compared with other nucleic acid sequences. Alternatively, if one or more cytosine (C) residues within a DNA sequence are unmethylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual, etc.), the sequence is considered to be demethylated or to have reduced methylation compared to the other nucleic acid sequence.

[0133] Herein, methylation level represents that one or more sites are in the ratio (or percentage, number, ratio, degree) of methylation state. The methylation level of a region (or a group of sites) is the mean of the methylation levels of all sites (or all sites in the group) in the region. Therefore, the methylation level rises or falls in a region and does not represent that the methylation levels of all methylation sites in the region rise or fall. It is known in the art that the method for detecting DNA methylation (such as simplified methylation sequencing) obtained results are converted into the process of methylation level. Methylation level can be determined by, for example, quantitative analysis of the amount of the complete DNA present after restrictive digestion with a methylation-sensitive restriction enzyme. In this example, if quantitative PCR is used to carry out quantitative analysis of the specific sequence in the DNA, the amount of template DNA approximately equals the control of the simulation treatment, which shows that the sequence is not highly methylated, and the amount of template is significantly less than the amount of template in the sample of the simulation treatment, which shows that there is methylated DNA in the sequence. Therefore, the methylation level in the above example can be used as a quantitative indicator of methylation state. When the methylation level of the sequence in the sample needs to be compared with the threshold level, this is particularly useful.

[0134] In one or more embodiments, the methylation level (e.g., Ct value) of the target marker is increased or decreased when compared to a reference level. When the methylation marker level (e.g., Ct value) meets a certain threshold, it is identified as having esophageal cancer, being at risk of developing esophageal cancer, or having progressed esophageal cancer. Exemplarily, the cutoff values ​​(thresholds) of the Ct values ​​in the plasma of each target point described herein are: BCAN is 40; LMX1B is 28; 15:89952513-89952639 is 40; RP11-17A4.2 is 28; SPAG6 is 30; CCDC181 is 27; RNF220 is 40; NKX2-5 is 38; SP9 is 38. A target Ct value below the corresponding cutoff value indicates that the patient has esophageal cancer, is at risk of developing esophageal cancer, or has progressed esophageal cancer, and a target Ct value above the corresponding cutoff value indicates that the patient does not have esophageal cancer, has a low risk of developing esophageal cancer, or has remitted esophageal cancer.

[0135] Alternatively, the methylation level of the target marker can be mathematically analyzed to obtain a score. For the sample being tested, when the score is greater than or less than a threshold, the result is determined to be positive, i.e., suffering from esophageal cancer, there is a risk of developing esophageal cancer, or esophageal cancer has progressed. Conventional mathematical analysis methods and the process of determining thresholds are known in the art, such as machine learning, which reflects the stability of the model by fitting the model in the training set and evaluating the model in the test set. For example, a non-parametric test based on rank sum is used to test the difference in methylation signals between the esophageal cancer group and the non-esophageal cancer group. For example, Lasso regression is used to penalize the regression coefficient of the site, thereby achieving feature selection of important sites. An exemplary method is a binary logistic regression mathematical model. For example, for differentially methylated markers, a binary logistic regression is constructed for the training group samples, and the accuracy, sensitivity and specificity of the test results and the area under the predictive characteristic curve (ROC) (AUC) are statistically analyzed using the model, and the test set sample prediction score is statistically analyzed. The method of constructing a binary logistic regression mathematical model is common knowledge in this field. The logistic regression mathematical model of n target points is as follows: Logit=a0+a1*Ct1+a2*Ct2......a n *Ct n

[0136] When n targets are combined, the Logit value can be obtained based on the Ct value of each target and the Logistic regression formula. The CT value and Logit value corresponding to the Youden index of a single target and multiple targets in the training set are used as cutoff values ​​to confirm the presence of esophageal tumors and assess the risk of esophageal tumor formation.

[0137] Any combination of the targets described herein, combined with a binary logistic regression mathematical model, can confirm the presence of esophageal tumors and assess the risk of esophageal tumor formation. As shown in Tables 5 and 7, the AUCs are higher than those of individual targets. In one or more embodiments, the combined target includes at least one or more of LMX1B, RP11-17A4.2, SPAG6, CCDC181, and RNF220. In some embodiments, the combined target includes at least SPAG6. In some embodiments, the combined target includes at least SPAG6 and CCDC181. In some embodiments, the combined target includes at least SPAG6, CCDC181, and LMX1B. In some embodiments, the combined target includes at least SPAG6, CCDC181, and 15:89952513-89952639. In some embodiments, the combined target includes at least SPAG6, CCDC181, and RNF220. In some embodiments, the combined target includes at least SPAG6, CCDC181, and NKX2-5. In some embodiments, the combined targets include at least SPAG6, CCDC181, and SP9. In some embodiments, the combined targets include at least SPAG6, CCDC181, LMX1B, and 15:89952513-89952639. In some embodiments, the combined targets include at least SPAG6, CCDC181, LMX1B, 15:89952513-89952639, RNF220, and NKX2-5.

[0138] The regression models and cutoff values ​​of some exemplary combined targets are shown in Table 8. A regression result below the corresponding cutoff value indicates that the patient has esophageal cancer, is at risk of developing esophageal cancer, or has progressed esophageal cancer. A target Ct value above the corresponding cutoff value indicates that the patient does not have esophageal cancer, has a low risk of developing esophageal cancer, or has had esophageal cancer remission:

[0139] The logistic regression model of SPAG6 and CCDC181 targets is: Logit = 9.03843 - 0.21044 * Ct T5 -0.06763*Ct T6 , the score cutoff value is 0.528;

[0140] The logistic regression model of 15:89952513-89952639, SPAG6 and CCDC181 targets is: Logit=12.91955-0.11469*Ct T3 -0.20035*Ct T5 -0.05918*Ct T6 , the score cutoff value is 0.565;

[0141] The logistic regression model of LMX1B, 15:89952513-89952639, SPAG6 and CCDC181 targets is: Logit=14.27820-0.07151*Ct T2 -0.11018*Ct T3 -0.17588*Ct T5 -0.05523*Ct T6 , the score cutoff value is 0.609;

[0142] The logistic regression model of LMX1B, 15:89952513-89952639, SPAG6, CCDC181 and SP9 targets is: Logit=23.23890-0.07585*Ct T2 -0.10088*Ct T3 -0.16924*Ct T5 -0.05087*Ct T6 -0.24058*Ct T9 , the score cutoff value is 0.580;

[0143] The logistic regression model of LMX1B, 15:89952513-89952639, SPAG6, CCDC181, RNF220 and SP9 targets was: Logit=23.390911-0.076888*Ct T2 -0.103440*Ct T3 -0.170765*Ct T5 -0.052027*Ct T6 +0.004896*Ct T7 -0.243005*Ct T9 , the score cutoff value is 0.574;

[0144] The logistic regression model of LMX1B, 15:89952513-89952639, SPAG6, CCDC181, RNF220, NKX2-5 and SP9 targets was: Logit=23.314123-0.077434*Ct T2 -0.105683*Ct T3 -0.171699*Ct T5 -0.052777*Ct T6 +0.003450*Ct T7 +0.006865*Ct T8 -0.242215*Ct T9 , the score cutoff value is 0.580;

[0145] The logistic regression model of BCAN, LMX1B, 15:89952513-89952639, SPAG6, CCDC181, RNF220, NKX2-5 and SP9 targets was: Logit = 22.9435820.008510*Ct T1 -0.077292*Ct T2 -0.105043*Ct T3 -0.171297*Ct T5 -0.052460*Ct T6 +0.002818*Ct T7 +0.006129*Ct T8 -0.241308*Ct T9 , the score cutoff value is 0.583;

[0146] The logistic regression model for all 9 targets is: Logit = 22.835815 + 0.009258 * Ct T1 -0.075563*Ct T2 -0.098871*Ct T3 -0.029325*Ct T4 -0.170868*Ct T5 -0.049466*Ct T6 +0.008217*Ct T7 +0.010682*Ct T8 -0.234492*Ct T9 , the score cutoff value is 0.529.

[0147] The methylation level / state of one or more CpG dinucleotide sequences within a DNA sequence (e.g., a target marker) can be determined by various analytical methods known in the art, preferably quantitative analysis methods. Exemplary analytical methods include: polymerase chain reaction, including real-time polymerase chain reaction, digital polymerase chain reaction, and PCR based on bisulfite conversion (e.g., methylation-specific PCR (MSP)); nucleic acid sequencing; whole genome methylation sequencing; simplified methylation sequencing; separation based on quality (e.g., electrophoresis, mass spectrometry); target capture (e.g., hybridization, microarray); methylation-sensitive restriction endonuclease analysis; methylation-sensitive high-resolution melting curve method; methylation profile analysis based on a chip; mass spectrometry; and fluorescence quantitative analysis. Herein, detection includes detecting any chain at a gene or site.

[0148] In some embodiments, quantitative analysis is performed by real-time PCR. Non-limiting examples of real-time PCR include HeavyMethyl™ PCR described by Cottrell et al., Nucl. Acids Res. 32:e10, 2003; MethyLight™ PCR described by Eads et al., Cancer Res. 59:2302-2306, 1999. TM PCR; Headloop PCR described by Rand et al., Nucl. Acids Res. 33:e 127, 2005.

[0149] As used herein, the term "HeavyMethyl™ PCR" refers to an art-recognized real-time PCR technique in which one or more non-extendable nucleic acid (e.g., oligonucleotide) blocks bind to bisulfite-treated nucleic acid in a methylation-specific manner (i.e., the blockers bind specifically to unmutated DNA under moderate to high stringency conditions). An amplification reaction is performed using one or more primers, which may optionally be methylation-specific, but are flanked by one or more blockers. In the presence of unmethylated nucleic acid (i.e., mutated DNA), the blockers bind and no PCR product is produced. The methylation level of nucleic acid in a sample is determined using a TaqMan™ assay essentially as described, for example, in Holland et al. Proc. Natl. Acad. Sci. USA 88:7276-7280 1991.

[0150] As used herein, the term "MethyLight TM "PCR" refers to a fluorescence-based real-time PCR technique recognized in the art, which uses a dual-labeled fluorescent oligonucleotide probe called TaqMan™ probe and is designed to hybridize to a CpG-rich sequence located between the forward and reverse amplification primers. The TaqMan TM The probe consists of a fluorescent "reporter moiety" and a "quencher moiety" covalently bound to the TaqMan TM The linker portion (e.g., phosphoramidite) to which the nucleotides of an oligonucleotide are attached. During PCR amplification, TaqMan TM The probe is cleaved by the 5' nuclease activity of Taq polymerase, generating a signal that is detected in real time during the PCR reaction. In this method, molecular beacons can be used as detectable probes, and the system is independent of the 5'-3' exonuclease activity of the DNA polymerase used (see Mhlanga and Malmberg, Methods 25: 463-471, 2001).

[0151] As used herein, the term "Headloop PCR" refers to an art-recognized real-time PCR method that selectively amplifies a target nucleic acid but suppresses the amplification of non-amplified target variants by extending the 3' stem-loop to form a hairpin structure that cannot provide further amplification template.

[0152] In some embodiments, the real-time PCR is a multiplex real-time PCR. As used herein, the term "multiplex" can refer to an assay or other analytical method that can simultaneously determine the presence and / or amount of multiple markers (e.g., multiple nucleic acid sequences) by using more than one marker, each marker having at least one different detection characteristic, such as a fluorescence characteristic (e.g., excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum) or fluorescence lifetime) or a unique nucleic acid or protein sequence characteristic.

[0153] In some embodiments, quantitative analysis is performed by nucleic acid sequencing. Exemplary methods of nucleic acid sequencing are known in the art, see, for example, Frommer et al. Proc. Natl. Acad. Sci. USA 89: 1827-1831 1992; Clark et al. Nucl. Acids Res. 22: 2990-2997 1994. For example, by comparing a sequence obtained from a sample not treated with bisulfite or a known nucleotide sequence of a target region with a sequence obtained from a sample treated with bisulfite, it is helpful to identify methylated cytosine in a DNA sequence. Compared to an untreated sample, a thymine residue detected at any cytosine site in a bisulfite-treated sample can be considered a mutation caused by bisulfite treatment, i.e., a methylated cytosine is present at that site.

[0154] Methods for sequencing DNA are known in the art and include, for example, the dideoxy chain termination method or the Maxam-Gilbert method (see Sambrook et al. Molecular Cloning A Laboratory Manual (2nd Ed. CSHP New York 1989)), pyrosequencing (see Uhlmann et al. Electrophoresis 23: 4072-4079 2002), solid-phase pyrosequencing (see Landegren et al. Genome Res. 8(8): 769-776 1998), solid-phase minisequencing (see, for example, Southern et al. Genomics 13: 1008-1017 1992), minisequencing using FRET (see, for example, Chen and Kwok Nucleic Acids Res. 25: 347-353 1997), sequencing by ligation, or ultra-deep sequencing (see Marguiles et al. Nature 437(7057): 376-80 (2005)).

[0155] In some embodiments, quantitative analysis is performed by mass-based separation (e.g., electrophoresis, mass spectrometry). For example, the presence of methylated cytosine residues can be detected by combined bisulfite restriction analysis (COBRA), essentially as described by Xiong and Laird Nucl. Acids Res. 25: 2532-2534 2001. This method takes advantage of the differences in restriction enzyme recognition sites between methylated and unmethylated nucleic acids following treatment with a compound that selectively mutates unmethylated cytosine residues (e.g., bisulfite). For example, the restriction endonuclease Taq1 cleaves the sequence TCGA, which will be TTGA after bisulfite treatment of unmethylated nucleic acid and, therefore, will not be cleaved. The digested and / or undigested nucleic acids are then detected using detection means known in the art, such as electrophoresis and / or mass spectrometry. As another example, after treatment with a compound that selectively mutates unmethylated cytosine residues, different techniques are used to detect nucleic acid differences in the amplified products based on differences in nucleotide sequence and / or secondary structure, such as methylation-specific single-strand conformation analysis (MS-SSCA) (Bianco et al. Hum. Mutat. 14: 289-293 1999), methylation-specific denaturing gradient gel electrophoresis (MS-DGGE) (Abrams and Stanton Methods Enzymol. 212: 71-74 1992), and methylation-specific denaturing high performance liquid chromatography (MS-DHPLC) (Deng et al. Chin. J. Cancer Res. 12: 171-191 2000).

[0156] In some embodiments, quantitative analysis is performed by target capture (e.g., hybridization, microarray). Suitable detection methods by hybridization are known in the art, such as Southern, dot blot, slot blot, or other nucleic acid hybridization formats (Kawai et al. Mol. Cell. Biol. 14: 7421-7427 1994; Gonzalgo et al. Cancer Res. 57: 594-599 1997). In some embodiments, the probes used for hybridization analysis are detectably labeled. In some embodiments, the nucleic acid-based probes used for hybridization analysis are unlabeled. Such unlabeled probes can be immobilized on a solid support such as a microarray and can hybridize to a detectably labeled target nucleic acid molecule. An example of a microarray is a methylation-specific microarray, which can be used to distinguish between sequences with converted cytosine residues and sequences with unconverted cytosine residues (see Adorjan et al. Nucl. Acids Res. 30: e21 2002). Hybridization-based analysis can also be used on nucleic acids treated with methylation-sensitive restriction enzymes. As another example, the methylation status of CpG dinucleotide sequences within a DNA sequence can be determined by oligonucleotide probes that hybridize with PCR amplification primers simultaneously with bisulfite-treated DNA (wherein the primers can be methylation-specific primers or standard primers).

[0157] In some embodiments, the quantitative analysis is performed in the presence of a detection reagent. As used herein, the term "detection reagent" is a reagent used to detect the presence, absence, or amount of nucleic acid in a quantitative analysis step. Various detection reagents known in the art can be used in this application. In some embodiments, the detection reagent is selected from the group consisting of fluorescent probes, intercalating dyes, chromophore-labeled probes, radioisotope-labeled probes, and biotin-labeled probes.

[0158] Preferably, herein, exemplary probe sequences, including the probe sequence of the control marker ACTB, are shown in Table 2.

[0159] The 5' end of the fluorescent probe is usually labeled with a fluorescent dye (such as FAM, HEX / VIC, TAMRA, Texas Red, or Cy5), and the 3' end is labeled with a quencher (such as BHQ1, BHQ2, BHQ3, DABCYL, or TAMRA). In some embodiments, the fluorescent dye of SEQ ID NO: 21 is VIC, and the quencher is BHQ1; the fluorescent dye of SEQ ID NO: 22 is VIC, and the quencher is BHQ1; the fluorescent dye of SEQ ID NO: 23 is CY5, and the quencher is BHQ3; the fluorescent dye of SEQ ID NO: 24 is VIC, and the quencher is BHQ1; the fluorescent dye of SEQ ID NO: 25 is CY5, and the quencher is BHQ3; the fluorescent dye of SEQ ID NO: 26 is FAM, and the quencher is BHQ1; the fluorescent dye of SEQ ID NO: 27 is CY5, and the quencher is BHQ3; the fluorescent dye of SEQ ID NO: 28 is CY5, and the quencher is BHQ3; the fluorescent dye of SEQ ID NO: 29 is VIC, and the quencher is BHQ1; and the fluorescent dye of SEQ ID NO: 30 is ROX, and the quencher is BHQ2.

[0160] Labeling can be accomplished by direct or indirect methods. Direct labeling involves labeling being directly (covalently or non-covalently) coupled to a reagent. Indirect labeling involves the combination (covalently or non-covalently) of a second reagent and a first reagent. The second reagent should specifically bind to the first reagent. The second reagent can be coupled with a suitable label and / or the second reagent is the target (receptor) of a third reagent that can be combined with the second reagent. Using secondary, tertiary or even higher order reagents can usually increase signal intensity. Suitable secondary and higher order reagents can include antibodies, secondary antibodies and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). Reagents or substrates can also be "labeled" by one or more tags as known in the art.

[0161] In some embodiments, the quantitative analysis comprises amplifying the treated DNA using a quantitative primer pair and a DNA polymerase. As used herein, the term "quantitative primer pair" refers to one or more primer pairs used in the quantitative analysis step. Preferably, the quantitative primer pair is capable of hybridizing to at least 9 consecutive nucleotides of the treated DNA under stringent conditions, moderately stringent conditions, or highly stringent conditions.

[0162] In some embodiments, the quantitative analysis includes determining the methylation level of one or more target markers based on the presence or level of multiple CpG dinucleotides, TpG dinucleotides, or CpA dinucleotides in the treated DNA. In some embodiments, the quantitative analysis includes determining the methylation level of cytosine residues based on the presence or level of one or more CpG dinucleotides in the treated DNA. In some embodiments, the quantitative analysis includes determining the methylation level of cytosine residues based on the presence or level of one or more TpG dinucleotides in the treated DNA. In some embodiments, the quantitative analysis includes determining the methylation level of cytosine residues based on the presence of CpA dinucleotides in the treated DNA.

[0163] In some embodiments, the quantitative analysis step is performed by separating the treated DNA product into a plurality of components. In some embodiments, the plurality of components are subjected to a plurality of different quantitative analysis tests, wherein different combinations of the treated DNA product (if present in the component) are quantitatively analyzed in one of the plurality of components. In some embodiments, control markers are quantitatively analyzed in each component.

[0164] In some embodiments, the methylation level of each target marker is quantified separately based on pre-amplified DNA using MSP (see Herman, supra), for example, by using one or more primers that specifically hybridize to unconverted sequences under moderate and / or high stringency conditions, generating amplification products only when the template contains methylated cytosine at a CpG site.

[0165] In some embodiments, the quantitative primer pair is designed to amplify at least a portion of the treated DNA product, i.e., the quantitative analysis is designed as nested PCR. Nested PCR is an improvement on PCR that aims to improve sensitivity and specificity. Nested PCR involves the use of two primer sets and two consecutive PCR reactions. A first round of amplification is performed to produce a first amplicon, and a second round of amplification is performed using a primer pair, in which one or both primers anneal to a site within the region defined by the initial primer pair, i.e., the second primer pair is considered to be "nested" in the first pair of primers. In this way, background amplification products from the first PCR reaction that do not contain the correct internal sequence are no longer further amplified in the second PCR reaction.

[0166] Typically, the PCR reaction solution contains Taq DNA polymerase, PCR buffer, primers, probe, dNTPs, Mg 2+ Preferably, the Taq DNA polymerase is a hot start Taq DNA polymerase. 2+The final concentration is 1.0-20.0 mM; the concentration of each primer is 100-500 nM; the concentration of each probe is 100-500 nM. Exemplary PCR reaction conditions are: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 56°C annealing and extension for 60 s, 40 cycles.

[0167] In some embodiments, the method of the present invention includes a pre-amplification step. One of the purposes of pre-amplifying the target marker is to increase the quantity of the target marker in the treated DNA. As used herein, the term "amplification" generally refers to any process that can cause the copy number of a molecule or a group of related molecules to increase. When "amplification" is used for polynucleotide molecules, it refers to the multiple copies of a polynucleotide molecule or a part of a polynucleotide molecule that are usually generated from a small amount of polynucleotide, wherein the amplified substance (amplicon, PCR amplicon) is usually detectable. The amplification of polynucleotides covers multiple chemical and enzymatic processes. The form of amplification includes generating multiple DNA copies from one or several copies of a template RNA or DNA molecule by polymerase chain reaction (reverse transcription PCR, PCR), chain displacement amplification (SDA) reaction, transcription-mediated amplification (TMA) reaction, nucleic acid sequence-based amplification (NASBA) reaction or ligase chain reaction (LCR).

[0168] Available pre-amplification primers can be used to pre-amplify the target marker in the treated DNA. As used herein, the term "primer" refers to a single-stranded oligonucleotide that can, under suitable conditions (e.g., buffer and temperature), in the presence of four different nucleoside triphosphates and a reagent (e.g., DNA polymerase) for polymerization, serve as the starting point for template-guided DNA synthesis. In any given case, the length of the primer depends on, for example, the intended use of the primer, and is typically within the range of 15 to 30 nucleotides. Short primer molecules typically require lower temperatures to form sufficiently stable hybrid complexes with the template. Primers do not necessarily reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template. A primer site is a region on the template that hybridizes with a primer. A primer pair is a set of primers comprising a 5' forward primer that hybridizes with the 5' end of the sequence to be amplified and a 3' reverse primer that hybridizes with the complementary strand at the 3' end of the sequence to be amplified. A person skilled in the art can design primers according to the marker to be amplified based on the common knowledge in the art (see, for example, PCR Primer: A Laboratory Manual Cold Spring Harbor Laboratories NY 1995). In addition, some software packages for designing optimal probes and / or primers for use in various analyses are publicly available, such as Primer 3 available from the Center for Genome Research Cambridge Mass. USA. Obviously, their potential uses should also be taken into consideration when designing probes or primers. For example, the primers designed for the purpose of the present invention may include at least one CpG site, or the amplification product obtained from the primers may include at least one CpG site. Tools for designing primers for detecting DNA methylation status are also known in the art, such as MethPrimer (Li LC and Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002 Nov; 18(11): 1427-31). In the present application, by using the pre-amplification primers as a primer pool, any target marker (each at least a portion of the target marker or a subregion of the target marker) in the treated DNA can be pre-amplified.

[0169] As used herein, the term "complementary" refers to hybridization or base pairing between nucleotides or nucleic acids, for example, between the two strands of a double-stranded DNA molecule, or between a primer binding site and an oligonucleotide primer on a single-stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are typically A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand, after optimal alignment and comparison, and with appropriate nucleotide insertions or deletions, pair with at least about 80% (typically at least about 90% to 95%, more preferably about 98% to 100%) of the nucleotides of the other strand. Alternatively, complementarity exists when an RNA or DNA strand hybridizes to its complementary sequence under selective hybridization conditions. Typically, selective hybridization occurs when there is at least about 65% (preferably at least about 75%, more preferably at least about 90%) complementarity over a stretch of at least 14 to 25 nucleotides. See M. Kanehisa Nucleic Acids Res. 12:203 (1984), incorporated herein by reference.

[0170] In some embodiments, the preamplification primer pool comprises at least one methylation-specific primer pair. In some embodiments, the preamplification primer pool comprises a plurality of methylation-specific primer pairs. In some embodiments, the preamplification step is performed by methylation-specific PCR ("MSP"), which is PCR using methylation-specific primers. This technology (i.e., MSP) has been described in Herman et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 1996 September 3; 93(18): 9821-6 and United States Patent No. 6265171.

[0171] As used herein, the term "methylation-specific primer pair" refers to a primer pair specifically designed to recognize CpG sites in order to exploit differences in methylation to amplify a specific target marker in the treated DNA. The primers act only on molecules with a specific methylation state or without a specific methylation state. For example, the primers can be oligonucleotides that, under stringent, moderately stringent, or highly stringent conditions, can hybridize specifically with a specific CpG site that is methylated in a methylation-specific manner, but cannot hybridize with a specific CpG site that is not methylated. Thus, the primers will specifically amplify a target marker that is methylated at a specific CpG site. For another example, the primers can be oligonucleotides that, under stringent, moderately stringent, or highly stringent conditions, can hybridize specifically with a specific CpG site that is unmethylated in a methylation-specific manner, but cannot hybridize with a specific CpG site that is methylated. Thus, the primers will specifically amplify a target marker that is not methylated at a specific CpG site. Therefore, in the present application, the use of methylation-specific primers in the pre-amplification of at least one target marker in the treated DNA can distinguish between methylated and unmethylated CpG sites. The methylation-specific primer pair of the present application comprises at least one primer that hybridizes to a bisulfite-treated CpG dinucleotide. Thus, the sequence of the primer specific for methylated DNA comprises at least one CpG dinucleotide, and the sequence of the primer specific for unmethylated DNA comprises a "T" at the C position of CpG and / or an "A" at the G position of CpG.

[0172] A methylation-specific primer pair typically comprises a forward primer and a reverse primer, each of which comprises an oligonucleotide sequence that hybridizes to at least 9 consecutive nucleotides of one of the target markers (or a subregion of the target marker) under stringent conditions, moderately stringent conditions, or highly stringent conditions, wherein the at least 9 consecutive nucleotides of one of the target markers (or a subregion of the target marker) comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG site.

[0173] As used herein, the term "hybridization" may refer to a process in which two single-stranded polynucleotides non-covalently associate to form a stable double-stranded polynucleotide. In one aspect, the resulting double-stranded polynucleotide may be a "hybrid" or "duplex." The salt concentration in "hybridization conditions" is typically less than about 1 M, often less than about 500 mM, and can be less than about 200 mM. A "hybridization buffer" includes a buffered saline solution, such as 5% SSPE, or other such buffers known in the art. The hybridization temperature can be as low as 5°C, but is typically above 22°C, more typically above about 30°C, and often above 37°C. Hybridization is typically performed under stringent conditions, meaning that a sequence will hybridize to its target sequence but not to other non-complementary sequences. Stringent conditions are sequence-dependent and vary in different circumstances. For example, longer fragments may require higher hybridization temperatures than shorter fragments for specific hybridization. Because other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, the presence of organic solvents, and the degree of base mismatching, the combination of parameters is more important than the absolute measurement of any one parameter alone. Typically, stringent conditions are selected to be about 5°C below the melting point (Tm) of the specific sequence at a specific ionic strength and pH. The Tm can be the temperature at which half of a population of double-stranded nucleic acid molecules is separated into single strands. Several equations for calculating the Tm of nucleic acids are well known in the art. As shown in standard references, a simple estimate of the Tm value can be calculated by the formula Tm = 81.5 + 0.41 (% G + C) when the nucleic acid is in 1 M NaCl aqueous solution (see, for example, Anderson and Young Quantitative Filter Hybridization in Nucleic Acid Hybridization (1985)). Other references (e.g., Allawi and SantaLucia Jr. Biochemistry 36:10581-94 (1997)) include alternative calculation methods that take into account structural and environmental as well as sequence characteristics when calculating Tm.

[0174] In general, the stability of hybrids is a function of ion concentration and temperature. Typically, hybridization reactions are performed under relatively low stringency conditions, followed by washing in wash solutions of varying, but higher, stringency. Exemplary stringent conditions include a pH of about 7.0 to about 8.3, a temperature of at least 25°C, and a sodium ion (or other salt) concentration of at least 0.01 M to no more than 1 M. For example, 5x SSPE (750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4) and a temperature of about 30°C are suitable for allele-specific hybridization, although the appropriate temperature depends on the length and / or GC content of the hybridization region. In one aspect, "hybridization stringency" for determining mismatch percentages can be as follows: 1) high stringency: 0.1x SSPE, 0.1% SDS, 65°C; 2) moderate stringency (also known as moderate stringency): 0.2x SSPE, 0.1% SDS, 50°C; and 3) low stringency: 1.0x SSPE, 0.1% SDS, 50°C. It should be understood that the same stringency can be achieved using alternative buffers, salts, and temperatures. For example, moderately stringent hybridization can refer to conditions that allow nucleic acid molecules (e.g., probes) to bind to complementary nucleic acid molecules. The hybridized nucleic acid molecules typically have at least 60% identity, including, for example, at least 70%, 75%, 80%, 85%, 90%, or 95% identity. Moderately stringent conditions can be conditions that achieve the same effect as the following conditions: hybridization at 42°C, 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS, followed by washing at 42°C, 0.2x SSPE, 0.2% SDS. Highly stringent conditions can be provided by, for example, hybridization at 42°C, 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS, followed by washing at 65°C, 0.1x SSPE, and 0.1% SDS. Low stringency hybridization can be achieved using the following conditions: 22°C, 10% formamide, 5x Denhardt's solution, 6x SSPE, 0.2% SDS hybridization, followed by washing in 1x SSPE, 0.2% SDS at 37°C. Denhardt's solution contains 1% polysucrose, 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, EDTA) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025M EDTA.Other suitable moderate and high stringency hybridization buffers and conditions are well known to those skilled in the art and are described, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Press Plainview NY (1989) and Ausubel et al. Short Protocols in Molecular Biology 4th ed. John Wiley & Sons (1999).

[0175] In some embodiments, the pre-amplification primer pool also includes a control primer pair for amplifying a control marker. Typically, a control marker is a nucleic acid with a known characteristic (e.g., a known sequence, a known copy number per cell), which is used to compare with an experimental target (e.g., a nucleic acid with an unknown concentration). The control can be an endogenous, preferably an invariant gene, to which the experimental nucleic acid or target nucleic acid in the analysis can be standardized. This type of control standardized because of differences between samples may occur in, for example, sample processing, analysis efficiency, etc., and allows accurate data comparison between samples, quantitative analysis of amplification efficiency and deviation.

[0176] In some embodiments, the pre-amplification primer pairs for the target markers described herein can be, for example, the corresponding primer pairs described in SEQ ID NOs: 1-18 herein.

[0177] V. Identification of Esophageal Cancer, Assessment of Risk of Developing Esophageal Cancer, and Assessment of Esophageal Cancer Progression

[0178] The present invention has discovered that the BCAN gene or genomic BCAN sequence, the LMX1B gene or genomic LMX1B sequence, the chromosome 15 sequence at positions 89952513-89952639, the RP11-17A4.2 (also known as PENK) gene or genomic RP11-17A4.2 (PENK) sequence, the SPAG6 gene or genomic SPAG6 sequence, the CCDC181 gene or genomic CCDC181 sequence, the RNF220 gene or genomic RNF220 sequence, the NKX2-5 gene or genomic NKX2-5 sequence, and the SP9 gene or genomic SP9 sequence. By detecting the methylation level of one or more of the target markers in a biological sample containing DNA from an individual, the presence of esophageal tumors can be confirmed, the risk of esophageal tumor formation can be assessed, and / or the progression of esophageal tumors can be assessed. Therefore, by comparing the detected methylation level with the corresponding reference level, esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in the subject can be identified or diagnosed based on the comparison results.

[0179] The term "comparison" as used herein refers to comparing the methylation level of the target marker obtained by the quantitative analysis step contained in the biological sample being tested with its corresponding reference level. It should be understood that the terms used herein refer to the comparison of corresponding parameters or values, for example, comparing the absolute amount with the absolute reference amount, comparing the concentration with the reference concentration, or comparing the intensity signal obtained from the sample being tested with the intensity signal of the same type of reference sample. The comparison can be performed manually or with the assistance of a computer. For computer-assisted comparisons, the value of the determined amount can be compared with the value of a suitable reference stored in a database by a computer program. The computer program can further evaluate the results of the comparison and automatically provide the desired evaluation in a suitable output format. Based on the comparison of the methylation level of each target marker in the quantitative analysis step with the corresponding reference level, individuals with esophageal cancer, individuals at risk of developing esophageal cancer, or individuals with esophageal cancer that is progressing can be identified.

[0180] The "reference level" can be derived from one or more reference samples, wherein the reference level is obtained from an experiment conducted in parallel with the experiment for detecting the sample of interest. Alternatively, the reference level can be obtained in a database comprising a collection of data, standards or levels from one or more reference samples or disease reference samples. In some embodiments, such a collection of data, standards or levels is standardized so that it can be used for comparison with data from one or more samples. "Standardization" is the process of converting raw measurement data into data that can be directly compared with other standardized data. Standardization is used to overcome analytical method-specific errors caused by different factors in different analytical methods, such as differences in sample loading, differences in binding efficiency, differences in detection sensitivity and other types of errors.

[0181] In some embodiments, the reference database includes methylation levels of target markers and / or other laboratory and clinical data from one or more reference samples. In some embodiments, the reference database includes methylation levels of target markers, each of which is normalized as a percentage of the methylation level of a control marker detected under the same conditions as the reference sample. In order to compare with such normalized methylation levels of the target marker, the methylation level of the target marker of the test sample is also measured and calculated as a percentage of the methylation level of the control marker detected under the same conditions as the test sample.

[0182] In some embodiments, a reference database is established by aggregating reference level data from reference samples obtained from healthy individuals and / or individuals without esophageal cancer. In some embodiments, a reference database is established by aggregating reference level data from reference samples obtained from individuals being treated for esophageal cancer.

[0183] Those skilled in the art can select the reference level according to the desired sensitivity and specificity. The means for determining a suitable reference level are known to those skilled in the art, for example, the reference level can be determined from data collected in clinical studies.

[0184] As used herein, "higher than the corresponding reference level" refers to the methylation level of the corresponding target marker or its target region being at least 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more of the corresponding reference level. As used herein, "lower than the corresponding reference level" refers to the methylation level of the corresponding target marker being 98%, 95%, 93%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or even lower of the corresponding reference level.

[0185] In addition to the above comparison, those skilled in the art can also determine an individual's esophageal cancer, risk of developing esophageal cancer, or progression of esophageal cancer based on various factors, such as age, gender, medical history, family history, symptoms, etc.

[0186] As used herein, the term "Ct value" refers to the cycle number at which the fluorescence of the PCR product can be detected above the background signal. The Ct value is inversely proportional to the number of target markers in the sample, that is, the lower the Ct value, the greater the number of target markers in the sample. In some embodiments, the smaller the CT value, the stronger the methylation signal. Exemplarily, the cutoff values ​​(thresholds) of the Ct values ​​of each target described herein in plasma are: BCAN is 40; LMX1B is 28; 15:89952513-89952639 is 40; RP11-17A4.2 is 28; SPAG6 is 30; CCDC181 is 27: RNF220 is 40; NKX2-5 is 38; SP9 is 38. Target Ct values ​​below the corresponding cutoff values ​​indicate that one has esophageal cancer, is at risk of developing esophageal cancer, or has progressed with esophageal cancer.

[0187] Herein, when detecting two or more genes or their target regions, binary logistic regression analysis is performed on the methylation levels (e.g., CT values) of the two or more genes or their target regions to obtain a fitting equation. In one or more embodiments, the two or more genes or their target regions include at least one or more of LMX1B, RP11-17A4.2, SPAG6, CCDC181, and RNF220. In some embodiments, the combined target includes at least SPAG6. In some embodiments, the combined target includes at least SPAG6 and CCDC181. In some embodiments, the combined target includes at least SPAG6, CCDC181, and LMX1B. In some embodiments, the combined target includes at least SPAG6, CCDC181, and 15:89952513-89952639. In some embodiments, the combined target includes at least SPAG6, CCDC181, and RNF220. In some embodiments, the combined target includes at least SPAG6, CCDC181, and NKX2-5. In some embodiments, the combined targets include at least SPAG6, CCDC181, and SP9. In some embodiments, the combined targets include at least SPAG6, CCDC181, LMX1B, and 15:89952513-89952639. In some embodiments, the combined targets include at least SPAG6, CCDC181, LMX1B, 15:89952513-89952639, RNF220, and NKX2-5. Table 8 shows some embodiments of more than two genes, wherein the regression results below the corresponding cutoff values ​​indicate that the patient has esophageal cancer, is at risk of developing esophageal cancer, or has progressed esophageal cancer.

[0188] In some embodiments, the methylation levels of the SPAG6 gene or the SPAG6 sequence of the genome or its target region (T5) and the CCDC181 gene or the CCDC181 sequence of the genome or its target region (T6) in the subject's plasma or esophageal tissue are detected, wherein the Ct values ​​of the two genes or their target regions are subjected to binary logistic regression analysis, and the fitting equation is score = 9.03843-0.21044*Ct T5 -0.06763*Ct T6 ; Among them, if the score is greater than 0.528, the result is judged to be positive, which means it is esophageal cancer or there is a risk of esophageal cancer.

[0189] In some embodiments, the methylation levels of the chr15:89952513-89952639 region (T3), the SPAG6 gene or the SPAG6 sequence of the genome or its target region (T5), and the CCDC181 gene or the CCDC181 sequence of the genome or its target region (T6) in the subject's plasma or esophageal tissue are detected, wherein the Ct values ​​of the three genes or their target regions are subjected to binary logistic regression analysis, and the fitting equation is score=12.91955-0.11469*Ct T3 -0.20035*Ct T5 -0.05918*Ct T6 ; Among them, if the score is greater than 0.565, the result is judged to be positive, which means it is esophageal cancer or there is a risk of esophageal cancer.

[0190] In some embodiments, the methylation levels of the LMX1B gene or the LMX1B sequence of the genome or its target region (T2), the chr15:89952513-89952639 region (T3), the SPAG6 gene or the SPAG6 sequence of the genome (T5) or its target region, and the CCDC181 gene or the CCDC181 sequence of the genome or its target region (T6) in the subject's plasma or esophageal tissue are detected, wherein the Ct values ​​of the four genes or their target regions are subjected to binary logistic regression analysis, and the fitting equation is score=14.27820-0.07151*Ct T2 -0.11018*Ct T3 -0.17588*Ct T5 -0.05523*Ct T6 ; Among them, if the score is greater than 0.609, the result is judged to be positive, which means it is esophageal cancer or there is a risk of esophageal cancer.

[0191] In some embodiments, the methylation levels of the LMX1B gene or the LMX1B sequence of the genome or its target region (T2), the chr15:89952513-89952639 region (T3), the SPAG6 gene or the SPAG6 sequence of the genome or its target region (T5), the CCDC181 gene or the CCDC181 sequence of the genome or its target region (T6), and the SP9 gene or the SP9 sequence of the genome or its target region (T9) in the plasma or esophageal tissue of the subject are detected, wherein the Ct values ​​of the five genes or their target regions are subjected to binary logistic regression analysis, and the fitting equation is score=23.23890-0.07585*Ct T2 -0.10088*Ct T3 -0.16924*Ct T5 -0.05087*Ct T6-0.24058*Ct T9 ; Among them, if the score is greater than 0.580, the result is judged to be positive, which means it is esophageal cancer or there is a risk of esophageal cancer.

[0192] In some embodiments, the methylation levels of the LMX1B gene or the LMX1B sequence of the genome or its target region (T2), the chr15:89952513-89952639 region (T3), the SPAG6 gene or the SPAG6 sequence of the genome or its target region (T5), the CCDC181 gene or the CCDC181 sequence of the genome or its target region (T6), the RNF220 gene or the RNF220 sequence of the genome or its target region (T7), and the SP9 gene or the SP9 sequence of the genome or its target region (T9) in the plasma or esophageal tissue of the subject are detected, wherein the Ct values ​​of the six genes or their target regions are subjected to binary logistic regression analysis, and the fitting equation is score=23.390911-0.076888*Ct T2 -0.103440*Ct T3 -0.170765*Ct T5 -0.052027*Ct T6 +0.004896*Ct T7 -0.243005*Ct T9 ; Among them, if the score is greater than 0.574, the result is judged to be positive, which means it is esophageal cancer or there is a risk of esophageal cancer.

[0193] In some embodiments, the methylation levels of the LMX1B gene or the LMX1B sequence of the genome or its target region (T2), the chr15:89952513-89952639 region (T3), the SPAG6 gene or the SPAG6 sequence of the genome or its target region (T5), the CCDC181 gene or the CCDC181 sequence of the genome or its target region (T6), the RNF220 gene or the RNF220 sequence of the genome or its target region (T7), the NKX2-5 gene or the NKX2-5 sequence of the genome or its target region (T8), and the SP9 gene or the SP9 sequence of the genome or its target region (T9) in the plasma or esophageal tissue of the subject are detected, wherein the Ct values ​​of the seven genes or their target regions are subjected to binary logistic regression analysis, and the fitting equation is score=23.314123-0.077434*Ct T2 -0.105683*Ct T3 -0.171699*Ct T5 -0.052777*Ct T6 +0.003450*Ct T7 +0.006865*CtT8 - 0.242215*Ct T9 ; Among them, if the score is greater than 0.580, the result is judged to be positive, which means it is esophageal cancer or there is a risk of esophageal cancer.

[0194] In some embodiments, the methylation levels of the BCAN gene or the BCAN sequence of the genome or its target region (T1), the LMX1B gene or the LMX1B sequence of the genome or its target region (T2), the chr15:89952513-89952639 region (T3), the SPAG6 gene or the SPAG6 sequence of the genome or its target region (T5), the CCDC181 gene or the CCDC181 sequence of the genome or its target region (T6), the RNF220 gene or the RNF220 sequence of the genome or its target region (T7), the NKX2-5 gene or the NKX2-5 sequence of the genome or its target region (T8), and the SP9 gene or the SP9 sequence of the genome or its target region (T9) in the plasma or esophageal tissue of the subject are detected, wherein the Ct values ​​of the eight genes or their target regions are subjected to binary logistic regression analysis, and the fitting equation is score=22.9435820.008510*Ct T1 -0.077292*Ct T2 -0.105043*Ct T3 -0.171297*Ct T5 -0.052460*Ct T6 +0.002818*Ct T7 +0.006129*Ct T8 -0.241308*Ct T9 ; Among them, if the score is greater than 0.583, the result is judged to be positive, which means it is esophageal cancer or there is a risk of esophageal cancer.

[0195] In some embodiments, the BCAN gene or genomic BCAN sequence or its target region (T1), the LMX1B gene or genomic LMX1B sequence or its target region (T2), the chr15:89952513-89952639 region (T3), the RP11-17A4.2 gene or genomic RP11-17A4.2 sequence or its target region (T4), the SPAG6 gene or genomic SPAG6 sequence or its target region (T5), the CCDC18 1 gene or genome or its target region (T6), RNF220 gene or genome or its target region (T7), NKX2-5 gene or genome or its target region (T8), and SP9 gene or genome or its target region (T9) methylation level, wherein the Ct values ​​of the nine genes or their target regions were subjected to binary logistic regression analysis, and the fitting equation was score = 22.835815 + 0.009258 * Ct T1 -0.075563*Ct T2 -0.098871*Ct T3 -0.029325*Ct T4 -0.170868*Ct T5 -0.049466*Ct T6 +0.008217*Ct T7 +0.010682*Ct T8 -0.234492*Ct T9 ; Among them, if the score is greater than 0.529, the result is judged to be positive, which means it is esophageal cancer or there is a risk of esophageal cancer.

[0196] VI. Compositions and Kits

[0197] The present invention provides a methylation detection kit / diagnostic kit, composition, and diagnostic reagent for diagnosing esophageal tumors, assessing the risk of esophageal tumor formation, and / or assessing the progression of esophageal tumors. The kit, composition, and diagnostic reagent may include primer molecules. Depending on the target marker or target region to be detected, the kit, composition, and diagnostic reagent may contain primer pairs capable of hybridizing to the target marker or target region to be detected under stringent conditions, moderately stringent conditions, or highly stringent conditions.

[0198] In some embodiments, the primer pairs in the kits, compositions, and diagnostic reagents can be selected from any one or more of the following primer pairs:

[0199] (1) A primer pair capable of amplifying a fragment of the BCAN gene amplified using SEQ ID NOs: 1 and 2 as primers;

[0200] (2) a primer pair capable of amplifying a fragment of the LMX1B gene amplified using SEQ ID NOs: 3 and 4 as primers;

[0201] (3) a primer pair capable of amplifying a fragment of the chr15:89952513-89952639 region obtained by using SEQ ID NOs:5 and 6 as primers;

[0202] (4) a primer pair capable of amplifying a fragment of the RP11-17A4.2 gene amplified using SEQ ID NOs: 7 and 8 as primers;

[0203] (5) a primer pair capable of amplifying a fragment of the SPAG6 gene amplified using SEQ ID NOs: 9 and 10 as primers;

[0204] (6) a primer pair capable of amplifying a fragment of the CCDC181 gene amplified using SEQ ID NOs: 11 and 12 as primers;

[0205] (7) a primer pair capable of amplifying a fragment of the RNF220 gene amplified using SEQ ID NOs: 13 and 14 as primers;

[0206] (8) a primer pair capable of amplifying a fragment of the NKX2-5 gene amplified using SEQ ID NOs: 15 and 16 as primers;

[0207] (9) A primer pair capable of amplifying a fragment of the SP9 gene amplified using SEQ ID NOs: 17 and 18 as primers.

[0208] In some embodiments, the primer pairs are selected from at least one or more of the following primer pairs: SEQ ID NOs: 1 and 2; SEQ ID NOs: 3 and 4; SEQ ID NOs: 5 and 6; SEQ ID NOs: 7 and 8; SEQ ID NOs: 9 and 10; SEQ ID NOs: 11 and 12; SEQ ID NOs: 13 and 14; SEQ ID NOs: 15 and 16; and SEQ ID NOs: 17 and 18.

[0209] In some embodiments, the primer pairs in the kits, compositions, and diagnostic reagents can be selected from the following primer pairs:

[0210] (1) a primer pair that at least can amplify a fragment of the SPAG6 gene amplified by using SEQ ID NOs: 9 and 10 as primers, such as at least SEQ ID NOs: 9 and 10;

[0211] (2) at least a primer pair that can amplify a fragment of the SPAG6 gene obtained by using SEQ ID NOs: 9 and 10 as primers, and a primer pair that can amplify a fragment of the CCDC181 gene obtained by using SEQ ID NOs: 11 and 12 as primers, such as at least SEQ ID NOs: 9-12;

[0212] (3) at least a primer pair capable of amplifying a fragment of the SPAG6 gene amplified by using SEQ ID NOs: 9 and 10 as primers, a primer pair capable of amplifying a fragment of the CCDC181 gene amplified by using SEQ ID NOs: 11 and 12 as primers, and a primer pair capable of amplifying a fragment of the LMX1B gene amplified by using SEQ ID NOs: 3 and 4 as primers, such as at least SEQ ID NOs: 3, 4, and 9-12;

[0213] (4) at least a primer pair capable of amplifying a fragment of the SPAG6 gene amplified by using SEQ ID NOs: 9 and 10 as primers, a primer pair capable of amplifying a fragment of the CCDC181 gene amplified by using SEQ ID NOs: 11 and 12 as primers, and a primer pair capable of amplifying a fragment of the chr15: 89952513-89952639 region amplified by using SEQ ID NOs: 5 and 6 as primers, such as at least SEQ ID NOs: 5-6 and 9-12;

[0214] (5) at least a primer pair capable of amplifying a fragment of the SPAG6 gene amplified by using SEQ ID NOs: 9 and 10 as primers, a primer pair capable of amplifying a fragment of the CCDC181 gene amplified by using SEQ ID NOs: 11 and 12 as primers, and a primer pair capable of amplifying a fragment of the RNF220 gene amplified by using SEQ ID NOs: 13 and 14 as primers, such as at least SEQ ID NOs: 9-14;

[0215] (6) at least a primer pair capable of amplifying a fragment of the SPAG6 gene obtained by using SEQ ID NOs: 9 and 10 as primers, a primer pair capable of amplifying a fragment of the CCDC181 gene obtained by using SEQ ID NOs: 11 and 12 as primers, and a primer pair capable of amplifying a fragment of the NKX2-5 gene obtained by using SEQ ID NOs: 15 and 16 as primers, such as at least SEQ ID NOs: 9-12 and 15-16;

[0216] (7) at least a primer pair capable of amplifying a fragment of the SPAG6 gene amplified by using SEQ ID NOs: 9 and 10 as primers, a primer pair capable of amplifying a fragment of the CCDC181 gene amplified by using SEQ ID NOs: 11 and 12 as primers, and a primer pair capable of amplifying a fragment of the SP9 gene amplified by using SEQ ID NOs: 17 and 18 as primers, such as at least SEQ ID NOs: 9-12 and 17-18;

[0217] (8) at least comprising a primer pair capable of amplifying a fragment of the SPAG6 gene amplified by using SEQ ID NOs: 9 and 10 as primers, a primer pair capable of amplifying a fragment of the CCDC181 gene amplified by using SEQ ID NOs: 11 and 12 as primers, a primer pair capable of amplifying a fragment of the LMX1B gene amplified by using SEQ ID NOs: 3 and 4 as primers, and a primer pair capable of amplifying a fragment of the chr15: 89952513-89952639 region amplified by using SEQ ID NOs: 5 and 6 as primers, such as comprising at least SEQ ID NOs: 3-6 and 9-12; and

[0218] (9) at least comprising a primer pair capable of amplifying a fragment of the SPAG6 gene amplified by using SEQ ID NOs: 9 and 10 as primers, a primer pair capable of amplifying a fragment of the CCDC181 gene amplified by using SEQ ID NOs: 11 and 12 as primers, a primer pair capable of amplifying a fragment of the LMX1B gene amplified by using SEQ ID NOs: 3 and 4 as primers, a primer pair capable of amplifying a fragment of the chr15: 89952513-89952639 region amplified by using SEQ ID NOs: 5 and 6 as primers, a primer pair capable of amplifying a fragment of the RNF220 gene amplified by using SEQ ID NOs: 13 and 14 as primers, and a primer pair capable of amplifying a fragment of the NKX2-5 gene amplified by using SEQ ID NOs: 15 and 16 as primers, such as at least comprising SEQ ID NOs: 3-6 and 9-16.

[0219] In some embodiments, the primer pairs contained in the kits, compositions and diagnostic reagents are primer pairs for amplifying target markers or target regions thereof selected from the group consisting of:

[0220] BCAN, LMX1B, chr15:89952513-89952639 region, RP11-17A4.2, SPAG6, CCDC181, RNF220, NKX2-5 or SP9 gene or their respective sequences in the genome or their respective target regions;

[0221] SPAG6 gene or genomic SPAG6 sequence or its target region (T5) and CCDC181 gene or genomic CCDC181 sequence or its target region (T6);

[0222] chr15: 89952513-89952639 region (T3), SPAG6 gene or genomic SPAG6 sequence or its target region (T5), and CCDC181 gene or genomic CCDC181 sequence or its target region (T6);

[0223] LMX1B gene or genomic LMX1B sequence or its target region (T2), chr15:89952513-89952639 region (T3), SPAG6 gene or genomic SPAG6 sequence (T5) or its target region, and CCDC181 gene or genomic CCDC181 sequence or its target region (T6);

[0224] LMX1B gene or genomic LMX1B sequence or its target region (T2), chr15:89952513-89952639 region (T3), SPAG6 gene or genomic SPAG6 sequence or its target region (T5), CCDC181 gene or genomic CCDC181 sequence or its target region (T6), and SP9 gene or genomic SP9 sequence or its target region (T9);

[0225] LMX1B gene or genomic LMX1B sequence or its target region (T2), chr15:89952513-89952639 region (T3), SPAG6 gene or genomic SPAG6 sequence or its target region (T5), CCDC181 gene or genomic CCDC181 sequence or its target region (T6), RNF220 gene or genomic RNF220 sequence or its target region (T7), and SP9 gene or genomic SP9 sequence or its target region (T9);

[0226] LMX1B gene or genomic LMX1B sequence or its target region (T2), chr15:89952513-89952639 region (T3), SPAG6 gene or genomic SPAG6 sequence or its target region (T5), CCDC181 gene or genomic CCDC181 sequence or its target region (T6), RNF220 gene or genomic RNF220 sequence or its target region (T7), NKX2-5 gene or genomic NKX2-5 sequence or its target region (T8) and SP9 gene or genomic SP9 sequence or its target region (T9);

[0227] BCAN gene or genomic BCAN sequence or target region thereof (T1), LMX1B gene or genomic LMX1B sequence or target region thereof (T2), chr15:89952513-89952639 region (T3), SPAG6 gene or genomic SPAG6 sequence or target region thereof (T5), CCDC181 gene or genomic CCDC181 sequence or target region thereof (T6), RNF220 gene or genomic RNF220 sequence or target region thereof (T7), NKX2-5 gene or genomic NKX2-5 sequence or target region thereof (T8) and SP9 gene or genomic SP9 sequence or target region thereof (T9); or

[0228] BCAN gene or BCAN sequence of the genome or its target region (T1), LMX1B gene or LMX1B sequence of the genome or its target region (T2), chr15:89952513-89952639 region (T3), RP11-17A4.2 gene or RP11-17A4.2 sequence of the genome or its target region (T4), SPAG6 gene or SPAG6 sequence of the genome or its target region (T5), CCDC181 gene or CCDC181 sequence of the genome or its target region (T6), RNF220 gene or RNF220 sequence of the genome or its target region (T7), NKX2-5 gene or NKX2-5 sequence of the genome or its target region (T8) and SP9 gene or SP9 sequence of the genome or its target region (T9).

[0229] In some preferred embodiments of these embodiments, the primer sequences of the primer pairs are:

[0230] SEQ ID NOs: 9 and 10, and SEQ ID NOs: 11 and 12;

[0231] SEQ ID NOs: 5 and 6, SEQ ID NOs: 9 and 10, and SEQ ID NOs: 11 and 12;

[0232] SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 9 and 10, and SEQ ID NOs: 11 and 12;

[0233] SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, and SEQ ID NOs: 17 and 18;

[0234] SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, and SEQ ID NOs: 17 and 18;

[0235] SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, and SEQ ID NOs: 17 and 18;

[0236] SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, and SEQ ID NOs: 17 and 18; or

[0237] SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, and SEQ ID NOs: 17 and 18.

[0238] The primers may also include primers for detecting an internal reference such as ACTB, for example, primers capable of amplifying a fragment of the ACTB gene amplified using SEQ ID NOs: 19 and 20. In some embodiments, the internal reference primer pair is SEQ ID NOs: 19 and 20.

[0239] In some embodiments, the primers are packaged in a single container or in separate containers. In some embodiments, the kit further comprises one or more blocking oligonucleotides.

[0240] In some embodiments, the kits, compositions and diagnostic reagents further comprise a detection agent. In some embodiments, the detection agent is selected from the group consisting of a fluorescent probe, an intercalating dye, a chromophore-labeled probe, a radioisotope-labeled probe and a biotin-labeled probe.

[0241] In preferred embodiments, the kits, compositions, and diagnostic reagents may contain fluorescent probes corresponding to the target marker or target region to be detected. The probe sequences in the kits or compositions may be arranged according to the single target or target combination described in any of the above embodiments. For example, the kit or composition includes at least a probe against SPAG6, at least a probe against SPAG6 and CCDC181, at least a probe against SPAG6, CCDC181 and LMX1B, at least a probe against SPAG6, CCDC181 and 15:89952513-89952639, at least a probe against SPAG6, CCDC181 and RNF220, at least a probe against SPAG6, CCDC181 and NKX2-5, at least a probe against SPAG6, CCDC181 and SP9, at least a probe against SPAG6, CCDC181, LMX1B and 15:89952513-89952639, or at least a probe against SPAG6, CCDC181, LMX1B, 15:89952513-89952639, RNF220 and NKX2-5.

[0242] Exemplarily, the fluorescent probe in the kit, composition and diagnostic reagent can be a fluorescent probe for detecting the following group of target markers or their target regions: BCAN, LMX1B, chr15:89952513-89952639 region, RP11-17A4.2, SPAG6, CCDC181, RNF220, NKX2-5 or SP9 gene or their respective sequences in the genome or their respective target regions; SPAG6 gene or genomic SPAG6 sequence or its target region (T5) and CCDC181 gene or genomic CCDC181 sequence or its target region (T6); chr15:89952513-89952639 region (T3) , SPAG6 gene or genome SPAG6 sequence or its target region (T5) and CCDC181 gene or genome CCDC181 sequence or its target region (T6); LMX1B gene or genome LMX1B sequence or its target region (T2), chr15: 89952513-89952639 region (T3), SPAG6 gene or genome SPAG6 sequence (T5) or its target region and CCDC181 gene or genome CCDC181 sequence or its target region (T6); LMX1B gene or genome LMX1B sequence or its target region (T2), chr15: 89952513-89952639 region ( T3), SPAG6 gene or genomic SPAG6 sequence or its target region (T5), CCDC181 gene or genomic CCDC181 sequence or its target region (T6) and SP9 gene or genomic SP9 sequence or its target region (T9); LMX1B gene or genomic LMX1B sequence or its target region (T2), chr15:89952513-89952639 region (T3), SPAG6 gene or genomic SPAG6 sequence or its target region (T5), CCDC181 gene or genomic CCDC181 sequence or its target region (T6), RNF220 gene or genomic RNF220 sequence or its target region (T7) and SP9 gene or genomic SP9 sequence or its target region (T9); LMX1B gene or genomic LMX1B sequence or its target region (T2), chr15:89952513-89952639 region (T3), SPAG6 gene or genomic SPAG6 sequence or its target region (T5), CCDC181 gene or genomic CCDC181 sequence or its target region (T6), RNF220 gene or genomic RNF220 sequence or its target region (T7), NKX2-5 gene or genomic NKX2-5 sequence or its target region (T8) and SP9 gene or genomic SP9 sequence or its target region (T9);BCAN gene or genomic BCAN sequence or its target region (T1), LMX1B gene or genomic LMX1B sequence or its target region (T2), chr15:89952513-89952639 region (T3), SPAG6 gene or genomic SPAG6 sequence or its target region (T5), CCDC181 gene or genomic CCDC181 sequence or its target region (T6), RNF220 gene or genomic RNF220 sequence or its target region (T7), NKX2-5 gene or genomic NKX2-5 sequence or its target region (T8) and SP9 gene or genomic SP9 sequence or its target region (T9); or BCAN gene or genomic BCAN sequence or its target region (T1), LMX1B gene or genomic LMX1B sequence or its target region (T2), chr 15:89952513-89952639 region (T3), RP11-17A4.2 gene or genomic RP11-17A4.2 sequence or its target region (T4), SPAG6 gene or genomic SPAG6 sequence or its target region (T5), CCDC181 gene or genomic CCDC181 sequence or its target region (T6), RNF220 gene or genomic RNF220 sequence or its target region (T7), NKX2-5 gene or genomic NKX2-5 sequence or its target region (T8), and SP9 gene or genomic SP9 sequence or its target region (T9).

[0243] More specifically, the nucleotide sequence of the fluorescent probe in the kit can be selected from the following group: any one of SEQ ID NO: 21-30; SEQ ID NO: 25, and SEQ ID NO: 26; SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 26; SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 26; SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 29; SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 29; SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29; SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29; or SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO: 29.

[0244] The probe also includes a probe for detecting an internal reference gene such as ACTB. In some embodiments, the nucleotide sequence of the probe for detecting the internal reference gene ACTB is shown in SEQ ID NO:30.

[0245] In some embodiments, the kit may further comprise a DNA polymerase and / or a container suitable for storing a biological sample obtained from an individual. In some embodiments, the kit further comprises instructions for use and / or explanations of the test results of the kit.

[0246] In some embodiments, the kits, compositions and diagnostic reagents may also include reagents for enzymatic or non-enzymatic conversion. In a preferred embodiment, the kit further includes a bisulfite reagent or a methylation sensitive restriction enzyme (MSRE). In some embodiments, the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite ions, and any combination thereof. In some embodiments, the bisulfite reagent is sodium bisulfite. In some embodiments, the MSRE is selected from the group consisting of HpaII enzyme, SalI enzyme, enzyme, ScrFI enzyme, BbeI enzyme, NotI enzyme, SmaI enzyme, XmaI enzyme, MboI enzyme, BstBI enzyme, ClaI enzyme, MluI enzyme, NaeI enzyme, NarI enzyme, PvuI enzyme, SacII enzyme, HhaI enzyme, and any combination thereof.

[0247] The kits, compositions and diagnostic reagents may also include a converted positive standard in which unmethylated cytosine is converted to a base that does not bind to guanine. The positive standard may be fully methylated.

[0248] The kit, composition and diagnostic reagent may further include a PCR reaction reagent. Preferably, the PCR reaction reagent includes Taq DNA polymerase, PCR buffer, dNTPs and Mg2+.

[0249] In some embodiments, the kits, compositions, and diagnostic reagents further comprise standard reagents that can be used to perform CpG position-specific methylation analysis, wherein the analysis comprises one or more of the following techniques: MS-SNuPE, MSP, MethyLight TM , HeavyMethylTM, COBRA, and nucleic acid sequencing.

[0250] In some embodiments, the kits, compositions and diagnostic reagents may contain additional reagents selected from the group consisting of buffers (e.g., restriction enzyme, PCR, storage or wash buffers), DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns) and DNA recovery components, etc.

[0251] The kit of the present application may further comprise one or more of the following components known in the art of DNA enrichment: a protein component that selectively binds to methylated DNA; a triplex-forming nucleic acid component, one or more linkers, optionally in a suitable solution; a substance or solution for ligation, such as a ligase, a buffer; a substance or solution for column chromatography; a substance or solution for immunologically based enrichment (such as immunoprecipitation); a substance or solution for nucleic acid amplification, such as PCR; a dye or dyes, if suitable for coupling agents, if suitable in solution; a substance or solution for hybridization; and / or a substance or solution for washing steps.

[0252] In other embodiments, the composition of the present invention comprises an isolated nucleic acid molecule selected from one or more of the following: (1) a fragment of the BCAN gene amplified using SEQ ID NOs: 1 and 2 or sequences having at least 90% identity thereto as primers; (2) a fragment of the LMX1B gene amplified using SEQ ID NOs: 3 and 4 or sequences having at least 90% identity thereto as primers; (3) a fragment of the 15:89952513-89952639 region amplified using SEQ ID NOs: 5 and 6 or sequences having at least 90% identity thereto as primers; (4) a fragment of the RP11-17A4.2 gene amplified using SEQ ID NOs: 7 and 8 or sequences having at least 90% identity thereto as primers; (5) a fragment of the SPAG6 gene amplified using SEQ ID NOs: 9 and 10 or sequences having at least 90% identity thereto as primers; (6) a fragment of the SEQ ID NOs: 10 and 11 or sequences having at least 90% identity thereto as primers; NO: 11 and 12 or sequences having at least 90% identity thereto as primers; (7) a fragment of the RNF220 gene amplified using SEQ ID NO: 13 and 14 or sequences having at least 90% identity thereto as primers; (8) a fragment of the NKX2-5 gene amplified using SEQ ID NO: 15 and 16 or sequences having at least 90% identity thereto as primers; (9) a fragment of the SP9 gene amplified using SEQ ID NO: 17 and 18 or sequences having at least 90% identity thereto as primers; optionally further comprising a fragment of the ACTB gene amplified using SEQ ID NO: 19 and 20 or sequences having at least 90% identity thereto as primers.

[0253] In some embodiments, the composition contains at least the SPAG6 gene target region, at least the SPAG6 gene target region and the CCDC18 gene target region, at least the SPAG6 gene target region, the CCDC181 gene target region and the LMX1B gene target region, at least the SPAG6 gene target region, the CCDC181 gene target region and 15:89952513-89952639, at least the SPAG6 gene target region, the CCDC181 gene target region and the RNF220 gene target region, at least the SPAG6 gene target region, the CCDC 181 gene target region and NKX2-5 gene target region, at least containing the SPAG6 gene target region, CCDC181 gene target region and SP9 gene target region, at least containing the SPAG6 gene target region, CCDC181 gene target region, LMX1B gene target region and 15:89952513-89952639, or at least containing the SPAG6 gene target region, CCDC181 gene target region, LMX1B gene target region, 15:89952513-89952639, RNF220 gene target region and NKX2-5 gene target region.

[0254] In a preferred embodiment, the composition contains: a SPAG6 gene target region and a CCDC181 target region; a chr15:89952513-89952639 region, a SPAG6 gene target region and a CCDC181 gene target region; a LMX1B gene target region, a chr15:89952513-89952639 region, a SPAG6 gene target region and a CCDC181 gene target region; a LMX1B gene target region, a chr15:89952513-89952639 region, a SPAG6 gene target region, a CCDC181 gene target region and a SP9 gene target region; a LMX1B gene target region, a chr15:89952513-89952639 region, a SPAG6 gene target region, a CCDC181 gene target region, a RNF220 gene target region and a SP9 gene target region; a LMX1B gene target region , chr15:89952513-89952639 region, SPAG6 gene target region, CCDC181 gene target region, RNF220 gene target region, NKX2-5 gene target region and SP9 gene target region; BCAN gene target region, LMX1B gene target region, chr15:89952513-89952639 region, SPAG6 gene target region, CCDC181 gene target region, RNF220 gene target region, NKX2-5 gene target region and SP9 gene target region; or BCAN gene target region, LMX1B gene target region, chr15:89952513-89952639 region, RP11-17A4.2 gene target region, SPAG6 gene target region, CCDC181 gene target region, RNF220 gene target region, NKX2-5 gene target region and SP9 gene target region.

[0255] The present application also includes a medium recording the sequence of the isolated nucleic acid molecule described herein and optionally its methylation information, and the medium is used to compare with gene methylation sequencing data to determine the presence, content and / or methylation level of the nucleic acid molecule. Preferably, the medium is a card printed with the sequence and optionally its methylation information, such as a paper, plastic, metal, or glass card. Preferably, the medium is a computer-readable medium storing the sequence and optionally its methylation information and a computer program, and when the computer program is executed by a processor, the following steps are implemented: comparing the methylation sequencing data of the sample with the sequence, thereby obtaining the presence, content and / or methylation level of the nucleic acid molecule containing the sequence in the sample.

[0256] The present application also includes a device for identifying esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer, the device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein when the processor executes the program, the following steps are implemented: (1) obtaining the methylation level of one or more target markers described herein or their target regions in a sample, (2) interpreting esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer based on the methylation level of (1). Preferably, the obtaining step is performed using any of the methods described in Section IV of the present application; preferably, the interpretation step is performed using any of the methods described in Section V of the present application.

[0257] VII. Application

[0258] The present application also provides the use of the primer pairs, combinations of primer pairs, and optional probes or probe combinations described herein in diagnosing esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer, as well as their use in preparing reagents or kits for diagnosing esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer.

[0259] Preferably, the kit is as described in any scheme in Section VI of this application. Preferably, the kit is used to implement the various steps and methods described in any one or more of Sections II, III, IV and V of this application.

[0260] The present application also provides the use of the isolated nucleic acid molecules described herein as detection targets in the diagnosis of esophageal cancer, the risk of esophageal cancer, or the progression of esophageal cancer.

[0261] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0262] Example 1: Comparison of DNA methylation abundance in esophageal cancer, paracancerous tissue, and buffy coat samples

[0263] DNA samples were obtained from the buffy coat layer of healthy individuals with normal esophageal abnormalities, as well as from cancerous and adjacent tissues of esophageal cancer patients (19 buffy coat samples, 20 adjacent tissues, and 20 adjacent tissues). Buffy coat DNA was chosen as a reference sample because the majority of plasma cell-free DNA is derived from DNA released after the buffy coat breaks down, and this background signal can serve as a baseline for the plasma cell-free DNA assay. Buffy coat DNA was extracted using the Qiagen QIAamp DNA Mini Kit, and tissue DNA was extracted using the Qiagen QIAamp DNA FFPE Tissue Kit, as specified in the manufacturer's instructions.

[0264] 20 ng of the DNA sample obtained in the above step was treated with a bisulfite reagent (D5031, ZYMO RESEARCH) to obtain converted DNA.

[0265] The fluorescent Ct value of the marker was obtained by fluorescence PCR detection. In the fluorescent PCR reaction system, the final concentration of each primer was 500nM, and the final concentration of each detection probe was 200nM. The PCR reaction system contained: 10μL of pre-amplification diluted product, 2.5μL of primer and probe premix containing the detection site; 12.5μL of PCR reagent ( Universal Probe qPCR Master Mix (NEB) was used. The primer sequences for each methylation marker and internal control are shown in Table 1, and the probe sequences are shown in Table 2. PCR reaction conditions were as follows: 95°C for 5 minutes; 95°C for 30 seconds, and 56°C for 60 seconds (for fluorescence collection), for 40 cycles. Fluorescence was detected in the corresponding fluorescence channels using the ABI 7500 Real-Time PCR System. Ct values ​​for samples obtained from the buffy coat, paracancerous tissue, and cancerous tissue were calculated and compared. Targets for which no amplification signal was detected had a Ct value of 40.

[0266] Table 1: Detection primer sequences

[0267] Table 2: Detection probe sequences

[0268] Table 3: Summary of sample test results

[0269] The results in Table 3 above show that the average Ct value detected in cancer tissue is lower, indicating a stronger methylation signal. The detection rate of methylation signals in cancer tissue is much higher than in the tunica buffy coat, also indicating a stronger methylation signal. In the majority of samples from the tunica buffy coat, no target methylation signals were detected. Therefore, these targets have the potential to be used in blood testing for esophageal cancer, demonstrating the feasibility and specificity of the selected target markers for tumor tissue.

[0270] Example 2: Comparison of methylation signals in plasma samples from esophageal cancer patients and esophageal control subjects

[0271] The plasma of 158 non-esophageal cancer individuals (as controls) and the preoperative plasma of 140 esophageal cancer patients (the proportions of I-IV were 25.7%, 25.0%, 44.3%, and 5.0%, respectively) were selected as training sets, and these samples were subjected to methylation detection.

[0272] The free DNA in plasma was extracted using a commercial cfDNA extraction kit cfDNA Isolation Kit (Magnetic Beads) (Cat. No.: EP120192).

[0273] The extracted extracellular free DNA was subjected to sulfite conversion using the commercial bisulfite conversion reagent MethylCodeTM Bisulfite Conversion Kit to obtain the converted DNA.

[0274] The converted DNA was used for pre-amplification. Specifically, a primer pool containing the methylation marker-specific primers shown in Table 1 and the internal reference ACTB primers (forward primer and reverse primer are shown in Table 1, and the probe is shown in Table 2) was used to perform PCR amplification using the converted DNA as a template. The final concentration of each primer was 100 nM. The PCR reaction system consisted of 10 μL of converted DNA, 2.5 μL of the premix containing the above primers; PCR reagents ( 12.5 μL of Universal Probe qPCR Master Mix (NEB) was added. PCR reaction conditions were as follows: 95°C for 5 minutes, followed by 15 cycles of 95°C for 30 seconds and 56°C for 60 seconds.

[0275] The obtained pre-amplification product was diluted 10 times and then used for fluorescence PCR detection. Fluorescence PCR was performed using the primers of each methylation marker shown in Table 1 and the probes shown in Table 2, and ACTB was used as a control. The final concentration of each primer was 500nM, and the final concentration of each probe was 200nM. The PCR reaction system contained: 10μL of pre-amplification diluted product, 2.5μL of primer and probe premix containing the detection site; PCR reagent ( Universal Probe qPCR Master Mix (NEB) 12.5μL.

[0276] Fluorescence PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 15 seconds, and 56°C for 40 seconds (for fluorescence acquisition), for 40 cycles. Fluorescence detection channels were selected based on the fluorescence modification of the different gene probes. The Ct value for targets where no amplification signal was detected was set to 40.

[0277] The Ct value of the test site was considered valid when the Ct value of the control ACTB was less than 20. The Ct values ​​of the test sites in esophageal cancer plasma and non-cancer control plasma were subjected to receiver operating curve analysis.

[0278] The areas under the receiver operating curves for the test sites are shown in Table 4 and Figure 1 below:

[0279] Table 4: Area under the receiver operating curve for the test sites

[0280] The results in the above table show that the above target markers have a high degree of discrimination for blood samples of esophageal cancer patients.

[0281] Binary logistic regression was used to classify the targets into 2-target combination, 3-target combination, 4-target combination, 5-target combination, and 6-target combination. The AUCs of representative combined logistic regressions are shown in Table 5 and Figure 3.

[0282] Table 5: Area under the receiver operating curve for target combination

[0283] The results in the table above show that the AUC of target combination detection is greater than that of a single target, proving that target combination can increase the discriminability of target markers for blood samples from esophageal cancer patients.

[0284] Example 3: Comparison of validation performance in plasma samples from esophageal cancer patients and esophageal control subjects

[0285] The plasma of 68 non-esophageal cancer individuals (as controls) and the preoperative plasma of 60 esophageal cancer patients (the proportions of I-IV were 21.7%, 28.3%, 41.7%, and 8.3%, respectively) were selected as validation sets, and methylation detection was performed on these samples.

[0286] The plasma DNA extraction method, sulfite conversion, pre-amplification and PCR reaction were the same as those in Example 2.

[0287] The area under the receiver operating characteristic curve (AOC) of each point was tested, and the results are shown in Table 6 and Figure 2 below.

[0288] Table 6: Area under the receiver operating curve for the test sites in the validation set

[0289] The multi-target combination logistic regression model of Example 2 was used to predict the population in the validation set. The validation set data are shown in Table 7 (see Table 7 and Figure 4), which further confirmed that the target combination can increase the discrimination of the target marker in blood samples of esophageal cancer patients.

[0290] Combinations of two, three, or even nine targets with the highest AUCs in both the training and test sets were selected. The Logit value was derived based on the Ct value of each target and the logistic regression formula. The Ct value and Logit value corresponding to the Youden Index for single targets and multi-target combinations in the training set were used as cutoffs to confirm the presence of esophageal tumors and assess the risk of esophageal tumor formation. The optimal combinations of single targets and different combinations are shown in Table 8, and the corresponding performance in the training and test sets is shown in Tables 9 and 10.

[0291] Table 7: Area under the receiver operating curve for target combinations in the validation set

[0292] Table 8: Cutoff values ​​for single target and multi-target combination

[0293] Table 9: Performance on the training set

[0294] Table 10: Performance on the test set

[0295] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to be within the scope of the appended claims and their equivalents.

Claims

1. Use of a reagent for evaluating the methylation state or level of a DNA region in a biological sample from an individual in the preparation of a diagnostic reagent or diagnostic kit for diagnosing esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in an individual; wherein: The DNA region is selected from: any one or more of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome.

2. Use of primer molecules and / or probe molecules in the preparation of a detection reagent or diagnostic kit for diagnosing individual esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer, and use of a device in the preparation of a diagnostic kit for diagnosing individual esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer; characterized in that: The primer molecule is identical to, complementary to, or hybridizes under stringent conditions to one or more target markers and comprises at least 9 consecutive nucleotides; The probe molecule hybridizes with the amplified product of one or more target markers under stringent conditions; The device is used to determine the methylation state or level of at least one CpG dinucleotide of one or more target markers; Wherein, the one or more target markers are selected from: BCAN gene or BCAN sequence of the genome, LMX1B gene or LMX1B sequence of the genome, sequence of chromosome 15 of the genome at positions 89952513-89952639, RP11-17A4.2 gene or RP11-17A4.2 sequence of the genome, SPAG6 gene or SPAG6 sequence of the genome, CCDC181 gene or CCDC181 sequence of the genome, RNF220 gene or RNF220 sequence of the genome, NKX2-5 gene or NKX2-5 sequence of the genome, SP9 gene or SP9 sequence of the genome, any one or more of them.

3. The use according to claim 2, characterized in that: The target marker comprises at least the SPAG6 sequence of the SPAG6 gene or genome, or at least the SPAG6 sequence of the SPAG6 gene or genome and the CCDC181 gene or genome CCDC181 sequence, or at least the SPAG6 sequence of the SPAG6 gene or genome, the CCDC181 sequence of the CCDC181 gene or genome and the LMX1B sequence of the LMX1B gene or genome, or at least the SPAG6 sequence of the SPAG6 gene or genome, the CCDC181 sequence of the CCDC181 gene or genome and the sequence of positions 89952513-89952639 of chromosome 15 of the genome, or at least the SPAG6 sequence of the SPAG6 gene or genome, the CCDC181 sequence of the CCDC181 gene or genome and the RNF220 sequence of the RNF220 gene or genome. sequence, or at least the SPAG6 sequence of the SPAG6 gene or genome, the CCDC181 gene or the CCDC181 sequence of the genome and the NKX2-5 gene or the NKX2-5 sequence of the genome, or at least the SPAG6 sequence of the SPAG6 gene or the genome, the CCDC181 sequence of the CCDC181 gene or the genome and the SP9 sequence of the SP9 gene or the genome, or at least the SPAG6 sequence of the SPAG6 gene or the genome, the CCDC181 sequence of the CCDC181 gene or the genome, the LMX1B sequence or the LMX1B sequence of the genome and the sequence at position 89952513-89952639 of chromosome 15 of the genome, or at least comprises the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence at position 89952513-89952639 of chromosome 15 of the genome, the RNF220 gene or the RNF220 sequence of the genome, and the NKX2-5 gene or the NKX2-5 sequence of the genome; or The target marker comprises the SPAG6 gene or the SPAG6 sequence of the genome and the NKX2-5 gene or the NKX2-5 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the SPAG6 gene or the SPAG6 sequence of the genome and the CCDC181 gene or the CCDC181 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the LMX1B gene or the LMX1B sequence of the genome and the SPAG6 gene or the SPAG6 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the sequence of chromosome 15, position 89952513-89952639, the SPAG6 gene or the SPAG6 sequence of the genome, and the CCDC181 gene or the CCDC181 sequence of the genome. Optionally, it further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, and the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome and the CCDC181 gene or the CCDC181 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises a sequence at position 89952513-89952639 of chromosome 15 of the genome, a SPAG6 gene or a SPAG6 sequence of the genome, a CCDC181 gene or a CCDC181 sequence of the genome, and a SP9 gene or a SP9 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or a BCAN sequence of the genome, the LMX1B gene or a LMX1B sequence of the genome, the RP11-17A4.2 gene or a RP11-17A4.2 sequence of the genome, the RNF220 gene or a RNF220 sequence of the genome, and the NKX2-5 gene or a NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence of chromosome 15 of the genome at position 89952513-89952639, the SPAG6 gene or the SPAG6 sequence of the genome, and the CCDC181 gene or the CCDC181 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the target marker is detected in the esophageal tissue or plasma from the individual. methylation levels; The target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, and the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome and the RNF220 gene or the RNF220 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence of position 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome and the NKX2-5 gene or the NKX2-5 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence of position 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target markers include LMX1B gene or LMX1B sequence of the genome, sequence of chromosome 15 of the genome at position 89952513-89952639, SPAG6 gene or SPAG6 sequence of the genome, CCDC181 gene or CCDC181 sequence of the genome, NKX2-5 gene or NKX2-5 sequence of the genome and SP9 gene. or the SP9 sequence of the genome, optionally further comprising one or more selected from the group consisting of the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, and the RNF220 gene or the RNF220 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the sequence at positions 89952513-89952639 of chromosome 15 of the genome, and the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence of position 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the BCAN gene or the BCAN sequence of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of chromosome 15 of the genome at positions 89952513-89952639, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of position 89952513-89952639 of chromosome 15 of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises one or more selected from the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of chromosome 15, position 89952513-89952639, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises the RNF220 gene or the RNF220 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target marker comprises the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome and the SP9 gene or the SP9 sequence of the genome, and optionally further comprises the NKX2-5 gene or the NKX2-5 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected; The target markers include the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome and the SP9 gene or the SP9 sequence of the genome; preferably, the methylation level of the target marker in the esophageal tissue or plasma from the individual is detected.

4. The use according to claim 2 or 3, characterized in that: The Hg19 coordinates of the genes or intergenic regions are as follows: BCAN gene, chr1: 156611182-156629324; LMX1B gene, chr9: 129376722-129463311; 15: 89952513-89952639, chr15: 89952513-89952639; RP11-17A4.2 gene, chr8: 57358366-57464626; SPAG6 gene Gene, chr10: 22634399-22743153; CCDC181 gene, chr1: 169364108-169429907; RNF220 gene, chr1: 44870866-45117396; NKX2-5 gene, chr5: 172659112-172662360; SP9 gene, chr2: 175199674-175203220.

5. The use according to claim 2 or 3, characterized in that: The one or more target markers are one or more target regions obtained by amplifying each gene or intergenic region using the following primers: Target region of the BCAN gene: a fragment of the BCAN gene amplified using the sequences shown in SEQ ID NOs: 1 and 2 or sequences having at least 90% identity to SEQ ID NOs: 1 and 2 as primers; Target region of the LMX1B gene: a fragment of the LMX1B gene amplified using the sequences shown in SEQ ID NOs: 3 and 4 or sequences having at least 90% identity to SEQ ID NOs: 3 and 4 as primers; 15: Target region of 89952513-89952639: a fragment amplified using the sequences shown in SEQ ID NOs: 5 and 6 or sequences having at least 90% identity to SEQ ID NOs: 5 and 6 as primers; Target region of RP11-17A4.2 gene: a fragment of RP11-17A4.2 gene amplified using the sequences shown in SEQ ID NOs: 7 and 8 or sequences having at least 90% identity with SEQ ID NOs: 7 and 8 as primers; Target region of the SPAG6 gene: a fragment of the SPAG6 gene amplified using the sequences shown in SEQ ID NOs: 9 and 10 or sequences having at least 90% identity to SEQ ID NOs: 9 and 10 as primers; Target region of the CCDC181 gene: a fragment of the CCDC181 gene amplified using the sequences shown in SEQ ID NOs: 11 and 12 or sequences having at least 90% identity to SEQ ID NOs: 11 and 12 as primers; Target region of the RNF220 gene: a fragment of the RNF220 gene amplified using the sequences shown in SEQ ID NOs: 13 and 14 or sequences having at least 90% identity to SEQ ID NOs: 13 and 14 as primers; Target region of NKX2-5 gene: a fragment of NKX2-5 gene amplified using the sequences shown in SEQ ID NOs: 15 and 16 or sequences having at least 90% identity to SEQ ID NOs: 15 and 16 as primers; Target region of SP9 gene: a fragment of SP9 gene amplified using the sequences shown in SEQ ID NOs: 17 and 18 or sequences having at least 90% identity with SEQ ID NOs: 17 and 18 as primers.

6. The use according to claim 5, characterized in that The Hg19 coordinates of the target regions of each gene or intergenic region are as follows: The target region of BCAN gene, chr1: 156611866-156611966; the target region of LMX1B gene, chr9: 129377872-129377947; the target region of 15: 89952513-89952639, chr15: 89952513-89952639; the target region of RP11-17A4.2 gene, chr8: 57358572-57358671; the target region of SPAG6 gene, chr10: 22634425-22634494; the target region of CCDC181 gene, chr1: 169396617-169396725; the target region of RNF220 gene, chr1: 44883356-44883454; the target region of NKX2-5 gene Target region, chr5: 172659753-172659839; target region of SP9 gene, chr2: 175202025-175202158.

7. The use according to any one of claims 1 to 6, characterized in that The primer molecules of each gene or intergenic region are respectively: BCAN: SEQ ID NOs: 1 and 2, or primer molecules that hybridize to the fragments amplified therefrom under stringent conditions; LMX1B: SEQ ID NOs: 3 and 4, or primer molecules that hybridize to the fragments amplified therefrom under stringent conditions; 15:89952513-89952639: SEQ ID NOs: 5 and 6, or primer molecules that hybridize to the fragments amplified therefrom under stringent conditions; RP11-17A4.2: SEQ ID NOs: 7 and 8, or primer molecules that hybridize to the fragments amplified therefrom under stringent conditions; SPAG6: SEQ ID NOs: 9 and 10, or primer molecules that hybridize to the fragments amplified therefrom under stringent conditions; CCDC181: SEQ ID NOs: 11 and 12, or primer molecules that hybridize to the fragments amplified therefrom under stringent conditions; RNF220: SEQ ID NOs: 13 and 14, or primer molecules that hybridize to the fragments amplified therefrom under stringent conditions; NKX2-5: SEQ ID NOs: 15 and 16, or primer molecules that hybridize to the fragments amplified therefrom under stringent conditions; SP9: SEQ ID NOs: 17 and 18, or primer molecules that hybridize to the fragments amplified therefrom under stringent conditions; The probe molecules of the amplified products of each gene or intergenic region are: BCAN: SEQ ID NO: 21 or a sequence at least 90% identical thereto; LMX1B: SEQ ID NO: 22 or a sequence at least 90% identical thereto; 15:89952513-89952639:SEQ ID NO:23 or a sequence having at least 90% identity thereof; RP11-17A4.2:SEQ ID NO:24 or a sequence having at least 90% identity thereof; SPAG6:SEQ ID NO:25 or a sequence having at least 90% identity thereof; CCDC181:SEQ ID NO:26 or a sequence having at least 90% identity thereof; RNF220:SEQ ID NO:27 or a sequence having at least 90% identity thereof; NKX2-5:SEQ ID NO:28 or a sequence having at least 90% identity thereof; SP9:SEQ ID NO:29 or a sequence having at least 90% identity thereof.

8. The use according to claim 2, characterized in that The detection reagent or diagnostic kit also includes primer molecules and / or probe molecules for detecting the internal reference gene ACTB; preferably, the primer molecules of the internal reference gene ACTB are sequences shown in SEQ ID NOs: 19 and 20 or sequences that hybridize under stringent conditions with a fragment of the ACTB gene amplified using SEQ ID NOs: 19 and 20 as primers, and the probe of the internal reference gene ACTB comprises the sequence shown in SEQ ID NO: 30 or a sequence that is at least 90% identical thereto.

9. A diagnostic reagent or diagnostic kit for detecting the methylation state or methylation level of at least one CpG dinucleotide of a target marker to diagnose esophageal cancer, the risk of esophageal cancer or the progression of esophageal cancer, comprising primer molecules and / or probe molecules; wherein, The primer molecule is identical to, complementary to or hybridizes to one or more target markers under stringent conditions and contains at least 9 consecutive nucleotides. The probe molecule hybridizes to the amplification product of one or more target markers under stringent conditions. The one or more target markers are selected from: any one or more of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome.

10. The diagnostic reagent or diagnostic kit according to claim 9, characterized in that The one or more target markers are as described in any one of claims 2-6; preferably, the primer molecules and probe molecules are as described in claim 7 or 8.

11. The diagnostic reagent or diagnostic kit according to claim 9 or 10, characterized in that: The diagnostic reagent or diagnostic kit also includes primer molecules and / or probe molecules for detecting the internal reference gene ACTB; preferably, the primer molecules of the internal reference gene ACTB are sequences shown in SEQ ID NOs: 19 and 20 or sequences that hybridize under stringent conditions with a fragment of the ACTB gene amplified using SEQ ID NOs: 19 and 20 as primers, and the probe of the internal reference gene ACTB comprises the sequence shown in SEQ ID NO: 30 or a sequence that is at least 90% identical thereto.

12. The diagnostic reagent or diagnostic kit according to claim 11, characterized in that The diagnostic kit also includes one or more substances selected from the following: PCR buffer, polymerase, dNTP, restriction endonuclease, enzyme cleavage buffer, fluorescent dye, fluorescence quencher, fluorescent reporter, exonuclease, alkaline phosphatase, internal standard, control, KCl, MgCl2 and (NH4)2SO4.

13. The diagnostic reagent or diagnostic kit according to claim 9, characterized in that Reagents for detecting DNA methylation also include reagents used in one or more of the following methods: PCR based on bisulfite conversion, DNA sequencing, methylation-sensitive restriction endonuclease analysis, fluorescence quantification, methylation-sensitive high-resolution melting curve method, chip-based methylation profiling and mass spectrometry.

14. The diagnostic reagent or diagnostic kit according to claim 13, characterized in that The reagent is selected from one or more of the following: bisulfite and its derivatives, fluorescent dyes, fluorescence quenchers, fluorescent reporters, internal standards and controls.

15. Use of at least one reagent or set of reagents that distinguishes between methylated and unmethylated CpG dinucleotides in at least one target region of genomic DNA in a kit for preparing a method for detecting and / or classifying esophageal cancer, risk of developing esophageal cancer, or progression of esophageal cancer in an individual, wherein the method comprises contacting genomic DNA isolated from a biological sample of the individual with the at least one reagent or set of reagents, wherein the target region is identical to or complementary to a sequence of at least 16 consecutive nucleotides of one or more target markers, wherein the consecutive nucleotides comprise at least one CpG dinucleotide sequence, thereby at least partially providing detection and / or classification of esophageal cancer, risk of developing esophageal cancer, or progression of esophageal cancer, wherein The one or more target markers are selected from: any one or more of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of chromosome 15 of the genome at positions 89952513-89952639, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome.

16. The use according to claim 15, characterized in that The one or more target markers are as described in any one of claims 2-6.

17. Use of one or more reagents that convert unmethylated cytosine base at position 5 to uracil or other bases detectably different from cytosine in hybridization properties, an amplification enzyme, and at least one primer comprising at least 9 consecutive nucleotides in a kit for the preparation of a method for detecting and / or classifying esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in an individual, wherein the method comprises: a) isolating genomic DNA from a biological sample of the individual; b) treating the genomic DNA or fragments thereof of a) with the one or more reagents; c) contacting the treated genomic DNA or the treated fragment thereof with the amplification enzyme and the at least one primer, the primer being identical to, complementary to, or hybridizing under stringent conditions to one or more target markers, wherein the treated genomic DNA or the fragment thereof is amplified to produce at least one amplification product or is not amplified; and d) determining the methylation state or level of at least one CpG dinucleotide of the one or more target markers, or a mean or value reflecting the average methylation state or level of multiple CpG dinucleotides of the one or more target markers, based on the presence or nature of the amplification product, thereby at least partially detecting and / or classifying esophageal cancer, the risk of developing esophageal cancer, or esophageal cancer progression in the individual; Wherein, the one or more target markers are selected from: BCAN gene or BCAN sequence of the genome, LMX1B gene or LMX1B sequence of the genome, sequence of chromosome 15 of the genome at positions 89952513-89952639, RP11-17A4.2 gene or RP11-17A4.2 sequence of the genome, SPAG6 gene or SPAG6 sequence of the genome, CCDC181 gene or CCDC181 sequence of the genome, RNF220 gene or RNF220 sequence of the genome, NKX2-5 gene or NKX2-5 sequence of the genome, SP9 gene or SP9 sequence of the genome, any one or more of them.

18. The use according to claim 17, characterized in that The one or more target markers are as described in any one of claims 2-6; preferably, the primer molecule is as described in claim 7 or 8.

19. The use according to claim 17, wherein in step b), the genomic DNA or fragments thereof is treated with a reagent selected from the group consisting of bisulfite, acid sulfite, pyrosulfite and a combination thereof.

20. The use according to claim 17, wherein in c), the contacting or amplification of nucleic acid molecules is carried out by using a thermostable DNA polymerase as the amplification enzyme, using a polymerase lacking 5'-3' exonuclease activity, using polymerase chain reaction and / or generating an amplification product with a detectable label.

21. The use of claim 17, wherein the contacting or amplifying in c) comprises using methylation-specific primers.

22. Use of one or more methylation-sensitive restriction enzymes and amplification enzymes and at least one primer comprising at least 9 consecutive nucleotides in the preparation of a kit for the method of detecting and / or classifying esophageal cancer, the risk of developing esophageal cancer or the progression of esophageal cancer in an individual, wherein: The primers are identical to, complementary to, or hybridize under stringent conditions to one or more target markers; the method comprises: a) isolating genomic DNA from a biological sample of the individual; b) digesting the genomic DNA or fragment thereof described in a) with the one or more methylation-sensitive restriction enzymes, and contacting the resulting digestion product with the amplification enzyme and the at least one primer; and c) determining the methylation state or level of at least one CpG dinucleotide of the one or more target markers based on the presence or nature of the amplification product, thereby at least partially detecting and / or classifying esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in the individual; Wherein, the one or more target markers are selected from: BCAN gene or BCAN sequence of the genome, LMX1B gene or LMX1B sequence of the genome, sequence of chromosome 15 of the genome at positions 89952513-89952639, RP11-17A4.2 gene or RP11-17A4.2 sequence of the genome, SPAG6 gene or SPAG6 sequence of the genome, CCDC181 gene or CCDC181 sequence of the genome, RNF220 gene or RNF220 sequence of the genome, NKX2-5 gene or NKX2-5 sequence of the genome, SP9 gene or SP9 sequence of the genome, any one or more of them.

23. The use according to claim 22, characterized in that The one or more target markers are as described in any one of claims 2-6; preferably, the primer molecule is as described in claim 7 or 8.

24. The use according to claim 22, characterized in that The presence or absence of the amplification product is determined by hybridizing at least one nucleic acid or peptide nucleic acid that is identical or complementary to a fragment of at least 16 bases long of a sequence selected from the one or more target markers.

25. Use of a treated nucleic acid derived from one or more target markers in the preparation of a kit for diagnosing esophageal cancer, the risk of developing esophageal cancer or the progression of esophageal cancer, wherein the treatment is suitable for converting at least one unmethylated cytosine base of the one or more target markers to uracil or other bases detectably different from cytosine in hybridization, and the one or more target markers are selected from: any one or more of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome.

26. The use according to claim 25, characterized in that The one or more target markers are as described in any one of claims 2-6.

27. A device for detecting and diagnosing esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer in an individual, the device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the following steps when executing the program: (1) obtaining the methylation level or methylation status of at least one CpG dinucleotide of one or more target markers in a sample, and (2) judging esophageal cancer, the risk of developing esophageal cancer, or the progression of esophageal cancer based on the methylation level or methylation status of (1); in, The one or more target markers are selected from: any one or more of the BCAN gene or the BCAN sequence of the genome, the LMX1B gene or the LMX1B sequence of the genome, the sequence of positions 89952513-89952639 of chromosome 15 of the genome, the RP11-17A4.2 gene or the RP11-17A4.2 sequence of the genome, the SPAG6 gene or the SPAG6 sequence of the genome, the CCDC181 gene or the CCDC181 sequence of the genome, the RNF220 gene or the RNF220 sequence of the genome, the NKX2-5 gene or the NKX2-5 sequence of the genome, and the SP9 gene or the SP9 sequence of the genome.

28. The device according to claim 27, characterized in that The one or more target markers are as described in any one of claims 2-6.

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