Esophageal cancer diagnosis kit based on multi-gene methylation level detection and application thereof

Abstract

This invention provides an esophageal cancer diagnostic kit based on multi-gene methylation level detection. It detects the methylation status of esophageal cancer gene methylation sites, including ZNF154, RNF126, C2ORF27A, OTOP2, TM4SF19, and P16, thereby enabling early screening of esophageal cancer samples. Furthermore, this kit, combined with digital PCR, offers advantages such as absolute quantification, high batch-to-batch stability, and high sensitivity, accurately distinguishing the relationship between sample gene methylation and esophageal cancer. Simultaneously, it employs fluorescently labeled probes for genotyping, demonstrating high specificity and applicability to esophageal cancer patients at different stages. Therefore, it provides a convenient, rapid, and accurate detection method for early screening, diagnosis, and prognosis of esophageal cancer, possessing significant clinical value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an esophageal cancer diagnostic kit based on the detection of multi-gene methylation levels and its application. Background Technology

[0002] Esophageal cancer ( Esophageal cancer, EC Cancer, a malignant tumor of the digestive tract, is the sixth leading cause of cancer-related deaths worldwide. China has an extremely high risk of developing the disease, ranking first in the world in new cases. Scopus Preview - Schilsky RLA, et al. NEJM. 2020;383:897-00 Typically, esophageal cancer patients in the early and middle stages only experience varying degrees of difficulty swallowing, easily missing the optimal time for treatment. By the time eating difficulties develop, most patients' esophageal tumors have progressed to an advanced stage, severely impacting clinical treatment and prognosis. Therefore, early screening for esophageal cancer is crucial, and effective and convenient examination methods can help improve the cure rate and quality of life for esophageal cancer patients.

[0003] Currently, the main methods for detecting esophageal cancer include tumor marker testing, esophageal cancer functional tests, imaging and endoscopic examinations, and cytological or histopathological examinations. Among these, endoscopy can effectively achieve early, highly sensitive detection; however, widespread endoscopic screening is challenging due to the high cost of equipment, the difficulty of operation, and low patient acceptance. Codipilly DC, et al. Gastrointest Endosc. 2018;88:413-426 Tumor markers, derived from the process of tumor development, can be collected from blood, saliva, excrement, epidermal cells, etc., and have an absolute advantage in detecting various tumors. However, there is still a lack of highly specific markers for esophageal cancer.

[0004] With the increasing maturity of biotechnology, precision medicine has become a call of the times. To achieve early screening and diagnosis of esophageal cancer in asymptomatic individuals, nucleic acid molecular testing may be a reasonable option. Like other tumors, alterations in multiple genes or epigenetic changes leading to changes in gene function are a direct cause of esophageal cell lesions and tumor development. Therefore, researchers are actively and extensively exploring the risk information contained in genes in esophageal cancer cases. Zang B, et al. Aging. 2020;12:3771-3790 ), and lncRNA ( Liu H, et al. Pathol Oncol Res. 2020;26:1029-1039 circRNA Shoda K, et al. Biomedicines. 2022;10:1643This research aims to understand the gene regulation of non-coding RNAs such as methylation, providing clinicians with information for esophageal cancer diagnosis, prognosis, and predictive treatment response. Gene methylation, as a major form of epigenetics, is closely related to carcinogenesis. Cancer risks include: promoter hypermethylation can silence tumor suppressor genes, while widespread genomic hypomethylation promotes oncogene activation and cell transformation. Currently, gene methylation has become a crucial detection target in early screening for other tumors. Therefore, in-depth research on esophageal cancer gene methylation will help solve the challenges of early esophageal cancer diagnosis. However, there are currently no documented applications of methylated gene combinations for esophageal cancer detection, both domestically and internationally. Therefore, developing a convenient, rapid, highly sensitive, and specific esophageal cancer detection method based on methylation combinations is urgently needed in this field. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide an esophageal cancer diagnostic kit based on the detection of multi-gene methylation levels, which has the characteristics of high sensitivity and specificity and convenient operation; at the same time, combined with digital PCR, it is suitable for esophageal cancer patients at different stages; thus, it provides a convenient, fast and accurate detection method for early screening, diagnosis and prognosis of esophageal cancer, and has important clinical value.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions:

[0007] An esophageal cancer diagnostic kit based on multi-gene methylation level detection, wherein the esophageal cancer gene methylation detection sites detected by the kit include ZNF154, RNF126, C2ORF27A, OTOP2, TM4SF19, and P16, the sequences of which are shown in SEQ ID NO. 1-12; and the kit includes a characteristic detection reagent, which includes a dye-based quantitative PCR premix and the following primer and probe combination:

[0008] 1) Primer and probe combinations for ZNF154 methylation detection, including Mut-specific primers as shown in SEQ ID NO.13, Mut-specific primers as shown in SEQ ID NO.14, Mut-specific probes as shown in SEQ ID NO.25, Wt-specific primers as shown in SEQ ID NO.31, Wt-specific primers as shown in SEQ ID NO.32, and Wt-specific probes as shown in SEQ ID NO.43;

[0009] 2) Primer and probe combinations for RNF126 methylation detection, including Mut-specific primers as shown in SEQ ID NO.15, Mut-specific primers as shown in SEQ ID NO.16, Mut-specific probes as shown in SEQ ID NO.26, Wt-specific primers as shown in SEQ ID NO.33, Wt-specific primers as shown in SEQ ID NO.34, and Wt-specific probes as shown in SEQ ID NO.44;

[0010] 3) Primer and probe combinations for C2ORF27A methylation detection, including Mut-specific primers as shown in SEQ ID NO.17, Mut-specific primers as shown in SEQ ID NO.18, Mut-specific probes as shown in SEQ ID NO.27, Wt-specific primers as shown in SEQ ID NO.35, Wt-specific primers as shown in SEQ ID NO.36, and Wt-specific probes as shown in SEQ ID NO.45;

[0011] 4) Primer and probe combinations for OTOP2 methylation detection, including Mut-specific primers as shown in SEQ ID NO.19, Mut-specific primers as shown in SEQ ID NO.20, Mut-specific probes as shown in SEQ ID NO.28, Wt-specific primers as shown in SEQ ID NO.37, Wt-specific primers as shown in SEQ ID NO.38, and Wt-specific probes as shown in SEQ ID NO.46;

[0012] 5) Primer and probe combinations for TM4SF19 methylation detection, including Mut-specific primers as shown in SEQ ID NO.21, Mut-specific primers as shown in SEQ ID NO.22, Mut-specific probes as shown in SEQ ID NO.29, Wt-specific primers as shown in SEQ ID NO.39, Wt-specific primers as shown in SEQ ID NO.40, and Wt-specific probes as shown in SEQ ID NO.47;

[0013] 6) Primer and probe combinations for P16 methylation detection, including Mut-specific primers as shown in SEQ ID NO.23, Mut-specific primers as shown in SEQ ID NO.24, Mut-specific probes as shown in SEQ ID NO.30, Wt-specific primers as shown in SEQ ID NO.41, Wt-specific primers as shown in SEQ ID NO.42, and Wt-specific probes as shown in SEQ ID NO.48.

[0014] Furthermore, the dye-based quantitative PCR premix is ​​a dye-based quantitative PCR premix from Baimeng Medical.

[0015] Furthermore, the test sample type of the kit is an ex vivo blood sample.

[0016] Furthermore, the 5' end of each of the specific probes contains a fluorescent group, which includes FAM and VIC; specifically, the fluorescent group of the Mut specific probe is FAM, and the fluorescent group of the Wt specific probe is VIC.

[0017] Furthermore, the 3' end of the specific probe contains a quenching group, which is MGB.

[0018] Furthermore, the reaction system of the kit is as follows: 1 μL of treated DNA (sulfurized template), 2.4 μL of 10 μM upstream and downstream primers, 0.75 μL of 10 μM Mut / Wt probe, 15 μL of dye-based quantitative PCR premix, and 8.45 μL of water are mixed to form a homogeneous 30 μL system.

[0019] Furthermore, the PCR reaction conditions for the kit are as follows (in microdroplets):

[0020]

[0021] An esophageal cancer detection method based on multi-gene methylation level detection, characterized by the following specific steps:

[0022] (1) Use magnetic bead method to extract cfDNA from the isolated blood sample to be tested, i.e., plasma, or extract genomic DNA from leukocytes;

[0023] (2) The cfDNA or genomic DNA to be detected is transformed using the bisulfite method;

[0024] (3) The methylation status of the DNA transformed in step (2) was detected by PCR technology.

[0025] Furthermore, in step (1), genomic DNA is extracted using the Cretaceous magnetic bead method universal DNA extraction kit; and cfDNA is extracted using the MIG magnetic bead method cell-free plasma DNA extraction kit.

[0026] Furthermore, the specific operation of step (2) is as follows:

[0027] Take 2000 ng of the cfDNA or genomic DNA extracted in step (1) and add it to the prepared pyrimidine conversion reaction solution. Mix well and centrifuge. Then, place it in a PCR instrument for reaction and purification by DNA adsorption. The PCR program is 98℃ / 10 min, 64℃ / 40 min, 98℃ / 5 min, 64℃ / 40 min, 98℃ / 5 min, 64℃ / 40 min, 4℃ / hold. The beneficial effects of this invention are:

[0028] (1) The kit of the present invention has the characteristics of high sensitivity and specificity and convenient operation;

[0029] (2) This kit uses DNA methylation abnormality as the detection target. DNA methylation abnormality usually occurs in the early stage of cancer and runs through the occurrence and development of cancer. Therefore, the detection of DNA methylation index can be used as an important indicator for early screening, diagnosis and prognosis assessment of esophageal cancer.

[0030] (3) The kit of the present invention is combined with digital PCR. Digital PCR has higher detection sensitivity and accuracy. It can efficiently and sensitively complete the PCR amplification of target nucleic acid fragments in micro-reaction units, effectively reduce the occurrence of false negatives, and obtain fluorescence signals for statistical analysis. Furthermore, the absolute quantification of the starting sample by digital PCR helps to obtain the relationship between methylation and esophageal cancer, thereby achieving effective screening for esophageal cancer. Attached Figure Description

[0031] The present invention will now be further described with reference to the accompanying drawings and embodiments.

[0032] Figure 1 This is the positive droplet ratio spectrum (FAM fluorescence) for identifying methylation mutations in Example 2 of the present invention;

[0033] Figure 2 This is the ratio spectrum (VIC fluorescence) of positive droplets that have not undergone methylation mutations in Example 2 of the present invention;

[0034] Figure 3 The ROC curve for ZNF154 detection of esophageal cancer in Example 2 of this invention;

[0035] Figure 4 The ROC curve for RNF126 detection of esophageal cancer in Example 2 of this invention;

[0036] Figure 5 The ROC curve for esophageal cancer detection using C2ORF27A in Example 2 of this invention;

[0037] Figure 6 This is the ROC curve for esophageal cancer detection using OTOP2 in Embodiment 2 of the present invention;

[0038] Figure 7 The ROC curve for esophageal cancer detection using TM4SF19 in Example 2 of this invention;

[0039] Figure 8 This is the ROC curve corresponding to the P16 detection of esophageal cancer in Embodiment 2 of the present invention;

[0040] Figure 9 The ROC curve for esophageal cancer is obtained by jointly detecting six genes in Example 2 of this invention.

[0041] Figure 10 This is the ROC curve corresponding to the early prediction of esophageal cancer in Embodiment 3 of the present invention;

[0042] Figure 11 This is the ROC curve corresponding to the mid-term prediction of esophageal cancer in Embodiment 3 of the present invention;

[0043] Figure 12 This is the ROC curve corresponding to the prediction of advanced esophageal cancer in Embodiment 3 of the present invention. Detailed Implementation

[0044] The present invention will be described in detail below with reference to specific embodiments, so that those skilled in the art can easily implement the invention based on the disclosure in the specification. The embodiments described in this invention are merely exemplary and are not intended to limit the invention. In addition, unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this technical field, and experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturer; and the samples used in this invention are all ex vivo samples.

[0045] Example 1: Screening of methylation detection sites and corresponding primers and probes

[0046] This invention uses TCGA data to obtain and analyze methylation data related to esophageal cancer, screens methylation sites with significant differences, and through multiple data filtering analyses, finally screens out six gene methylation detection sites: ZNF154, RNF126, C2ORF27A, OTOP2, TM4SF19, and P16. The methylated sequences of these six detection sites are shown in SEQ ID NO. 1-6, and the unmethylated sequences are shown in SEQ ID NO. 7-12.

[0047] Based on the nucleic acid sequences of ZNF154, RNF126, C2ORF27A, OTOP2, TM4SF19, and P16, the applicant repeatedly designed and refined the corresponding specific primer and probe combinations, which were then sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The specific sequences are as follows:

[0048] 1) Primer and probe combinations for ZNF154 methylation detection, including Mut-specific primer pairs (as shown in SEQ ID NO: 13, 14): upstream primer 5'-AGCGTCGGATGGGTTTAC-3', downstream primer 5'-ACTAAACCGAAAACGACG-3'; Mut-specific probe (as shown in SEQ ID NO: 25) 5'-TTATGCAGACGTTCGT-3'; Wt-specific primer pairs (as shown in SEQ ID NO: 31, 32): upstream primer 5'-AGAGTGGTGAATTAGGGTTTATG-3', downstream primer 5'-AAGCAATAACAGAGACTACATT-3'; Wt-specific probe (as shown in SEQ ID NO: 43) 5'-ATGTGTCATGATGTTTGTGG-3';

[0049] 2) Primer and probe combinations for RNF126 methylation detection, including Mut-specific primer pairs (as shown in SEQ ID NO. 15 and 16): upstream primer 5'-GATGAGCCGTTCAGTC-3', downstream primer 5'-CTTCACACTACGCCGCA-3'; Mut-specific probe (as shown in SEQ ID NO. 26) 5'-TATCGAGATAGTTGTATTCG-3'; Wt-specific primer pairs (as shown in SEQ ID NO. 33 and 34): upstream primer 5'-GAGTTGTCTGTATGGAGTATGT-3', downstream primer 5'-AAGTTTAATAATCTCAAACTATCCA-3'; Wt-specific probe (as shown in SEQ ID NO. 44) 5'-TTTATGTTGTTGTAAGGGTAT-3';

[0050] 3) Primer and probe combinations for C2ORF27A methylation detection, including Mut-specific primer pairs (as shown in SEQ ID NO. 17 and 18): upstream primer 5'-ACGATGGTCTGCGTAGG-3', downstream primer 5'-TAGGACGTCGACAGACG-3'; Mut-specific probe (as shown in SEQ ID NO. 27) 5'-ATTAGCGGTATATATCGTA-3'; Wt-specific primer pairs (as shown in SEQ ID NO. 35 and 36): upstream primer 5'-GTTGGCTAGTCTTTATGAGGTAT-3', downstream primer 5'-AATAACATAAGAACGAAGTC-3'; Wt-specific probe (as shown in SEQ ID NO. 45) 5'-TGGTGTGTGTTGGTTATGT-3';

[0051] 4) Primer and probe combinations for OTOP2 methylation detection, including Mut-specific primer pairs (as shown in SEQ ID NO. 19 and 20): upstream primer 5'-GTTCGTTATTATGCGTCGA-3', downstream primer 5'-GCAAAAACAACGCACGATTC-3'; Mut-specific probe (as shown in SEQ ID NO. 28) 5'-CGATTGTCTATGCAGTGA-3'; Wt-specific primer pairs (as shown in SEQ ID NO. 37 and 38): upstream primer 5'-TGTGATTTGATTAGTGAGTGGAGTTT-3', downstream primer 5'-TACCATCACACAAACAAGATACAT-3'; Wt-specific probe (as shown in SEQ ID NO. 46) 5'-ATGTGTATTGATGTGTGTATTATGT-3';

[0052] 5) Primer and probe combinations for TM4SF19 methylation detection, including Mut-specific primer pairs (as shown in SEQ ID NO. 21 and 22): upstream primer 5'-TTAGAGTCGAGATTTCTCG-3', downstream primer 5'-GCTCTCGTGCACCCG-3'; Mut-specific probe (as shown in SEQ ID NO. 29) 5'-AGGTGATTCTTACTGTGTG-3'; Wt-specific primer pairs (as shown in SEQ ID NO: 39 and 40): upstream primer 5'-ATTAATGAGTTGAAATTGTTTGT-3', downstream primer 5'-AAGAGTTCACCGTTGGACA-3'; Wt-specific probe (as shown in SEQ ID NO. 47) 5'-AGTAGGAGAGTGAAGGTGTT-3';

[0053] 6) Primer and probe combinations for P16 methylation detection, including Mut-specific primer pairs (as shown in SEQ ID NO. 23 and 24): upstream primer 5'-TCGTTAGTTATAGTATTTTCAG-3', downstream primer 5'-CTATTCTATCTCCGATACTAC-3'; Mut-specific probe (as shown in SEQ ID NO. 30) 5'-CGTATACGGGCGTCTTTT-3'; Wt-specific primer pairs (as shown in SEQ ID NO: 41 and 42): upstream primer 5'-TTGGAGGTAATCGTATTTTTTATTG-3', downstream primer 5'-TAACATTATCTGGCACATTGTAACA-3'; Wt-specific probe (as shown in SEQ ID NO. 48) 5'-TTAGTGTATTGTGTATTAATG-3'.

[0054] Mut-specific primers are primers that recognize methylation mutations in the corresponding gene, Wt-specific primers are primers that recognize the non-methylation mutations in the corresponding gene, Mut-specific probes are probes that recognize methylation mutations in the corresponding gene, and Wt-specific probes are probes that recognize the non-methylation mutations in the corresponding gene.

[0055] The kit of the present invention includes a feature detection reagent, which includes a dye-based quantitative PCR premix and primer and probe combinations corresponding to the six gene methylation detection sites mentioned above; wherein the dye-based quantitative PCR premix can be selected from the dye-based quantitative PCR premix from Baimeng Medical.

[0056] It should be noted that the probes of this invention are designed based on the SNP genotyping principle. Only probes that correctly bind to the mutation site will be cleaved by the 5' exonuclease activity of DNA polymerase, thereby releasing a fluorescent signal. This, together with specific primers, increases the specificity of multi-site mutation recognition. Simultaneously, the partial introduction of MGBs increases the stability of probe-template binding, thereby shortening the probe length and improving binding specificity. The following principles must be followed when designing Mut-specific and Wt-specific probes in this invention:

[0057] a) The probe Tm value is between 65-67 ℃, and the Tm value between Mut-specific probe and Wt-specific probe at the same methylation detection site differs by less than 1 ℃;

[0058] b) The probe length is between 13-25 bp;

[0059] c) Place the mutation site at 1 / 3 of the probe, or near the 3' end of the probe but not at the last two bases;

[0060] d) The 5' base of the probe cannot be G;

[0061] e) The distance between the 5' end of the probe and the 3' end of the primer in the same direction should be controlled within the range of 1-20 bp from near to far;

[0062] f) The probe binds to the target fragment with strong specificity and high efficiency.

[0063] Example 2: Verification of Sensitivity and Specificity

[0064] In this embodiment, 300 blood samples from esophageal cancer patients and 300 normal blood samples were used to establish a methylation assessment model.

[0065] 1. Sample preservation: Blood samples are collected by medical staff in anticoagulant preservation tubes and transported to the laboratory at room temperature. The time from collection to processing is controlled within 3 days.

[0066] 2. DNA Preparation: Blood samples from relevant cancers were obtained; genomic DNA was extracted using a Cretaceous magnetic bead universal DNA extraction kit. 200 μL of the blood white film layer was added to 300 μL of lysis buffer (Buffer AL), incubated at 58°C for 15 min, then 300 μL of isopropanol and 15 μL of extraction buffer (MB Mix) were added and mixed thoroughly, followed by purification. Cell-free DNA (cfDNA) was extracted using a MIKI magnetic bead plasma cell-free DNA extraction kit. 200 μL of proteinase K, 2 ml of lysis buffer, and 125 μL of magnetic bead suspension were added sequentially to 2 ml of plasma and incubated for 10 min. After washing with 2 ml of washing buffer, the DNA was dried at 56°C for 8 min and finally collected using 50 μL of elution buffer. The concentration was then determined using Qbuit to obtain the DNA content.

[0067] 3. Sulfite modification: Take 2000 ng of the prepared cfDNA or genomic DNA and put it into the prepared pyrimidine conversion reaction solution (CT Conversion Mix). Mix well and centrifuge briefly. Then put it into a PCR instrument for reaction. After the PCR reaction is completed, purify the transformation product through EpiArt DNA Column to obtain the treated DNA, i.e., the sulfite template. The PCR instrument reaction program is 98℃ / 10 min, 64℃ / 40 min, 98℃ / 5 min, 64℃ / 40 min, 98℃ / 5 min, 64℃ / 40 min, 4℃ / hold.

[0068] 4. Digital PCR: First, prepare a 30 μL reaction system including 1 μL of the sulfurized template described above, 2.4 μL of 10 μM upstream and downstream primers, 0.75 μL of 10 μM Mut / Wt probe, and 15 μL of dye-based quantitative PCR premix (SYBR). Prepare 8.45 μL of water (GreenMix); then generate reaction droplets using a droplet generator, start the sample preparation instrument, and perform a self-test; after the self-test is complete, open the instrument cover, first place the 8-tube array (provided by the kit) into the corresponding position on the instrument, insert the droplet generation chip into the matching generation chip mechanical clamp, press down the clamp cover to fix the chip, add 30 μL of the sample to be tested into the water well, and add 180 μL of droplet generation oil into the oil well, then cover the water well and oil well of the chip with the droplet generation chip sealing gasket; place the clamp containing the droplet generation chip into the corresponding position on the instrument; then press down the handle to fix it, and close the instrument cover; operate the instrument to generate droplets; after the droplets are generated, remove the clamp, and close the 8-tube array containing the droplets with the 8-tube array cap, ready for use; the droplet preparation step is generally completed within 5 minutes. Afterwards, place the 8-tube array containing the prepared droplet reaction solution in a PCR instrument for reaction, and the PCR reaction conditions are as follows:

[0069]

[0070] It should be noted that the annealing temperature is selected between 56-62℃ based on the TM value of each primer combination.

[0071] 5. Droplet Reading and Signal Analysis: Place the 8-tube strip containing the completed digital PCR reaction into the corresponding slot of the droplet reading chip, and add an appropriate amount of droplet detection oil to the designated well. After the reading chip is assembled, place it in the chip reader. Each sample requires approximately 5 minutes to read the results. Finally, obtain the positive droplet ratio graph (e.g., ...). Figure 1 and Figure 2 (as shown) Figure 1 The four sections are, in order: background wells, 100% unmethylated wells at multiple sites, 100% methylated wells at multiple sites, and droplet status of sample wells. Figure 2 The four sections are, in order, background wells, 100% methylated wells at multiple sites, 100% unmethylated wells at multiple sites, and droplet status of sample wells; (Refer to...) Figure 1 and Figure 2 It can be seen that the number of positive and negative droplets in the sample wells can be obtained under the control of DNA plasmids with 100% methylation at multiple sites and non-100% methylation at multiple sites.

[0072] 6. Plotting the corresponding ROC curves: In the sample detection, ROC curves were plotted with FPR=FP / (FP+TN) as the x-axis and TPR=TP / (TP+FN) as the y-axis (where TP represents the number of samples that were both predicted and actually negative; FN represents the number of samples that were predicted positive but actually negative; FP represents the number of samples that were predicted negative but actually positive; TN represents the number of samples that were both predicted and actually positive). The ROC curves for the detection of esophageal cancer using ZNF154, RNF126, C2ORF27A, OTOP2, TM4SF19, P16, and these six genes are as follows: Figure 4-9 As shown.

[0073] 7. Results Analysis: Refer to Figures 4 to 9 It can be seen that when using a single gene to detect esophageal cancer, the AUC value of the ROC curve for ZNF154 is 0.949, for RNF126 it is 0.907, for C2ORF27A it is 0.912, for OTOP2 it is 0.906, for TM4SF19 it is 0.911, and for P16 it is 0.891; while when using 6 genes together to detect esophageal cancer, the corresponding AUC value of the ROC curve is 0.96.

[0074] Amplification positivity was determined based on a Ct value less than the Cutoff value. The Cutoff values ​​for these six genes were 38.12, 37.77, 40.68, 38.26, 42.92, and 38.47, respectively. Methylation levels were then calculated based on the positive droplet ratio, and the degree of methylation was determined. A methylation level higher than 50% for the detected gene was considered strong methylation. If the detected gene showed strong methylation, the sample was considered a positive sample for esophageal cancer; otherwise, it was considered a negative sample for esophageal cancer. Ultimately, with the combined detection of these six genes according to this standard, the detection sensitivity for esophageal cancer increased to 91%, and the detection specificity reached 99%. The detection results of the samples in this embodiment are shown in Table 1 below.

[0075] Table 1. Detection results of esophageal cancer and normal samples 8.

[0077] 9. The simultaneous detection of ZNF154, RNF126, C2ORF27A, OTOP2, TM4SF19, and P16 in real time to analyze the DNA of blood samples fully demonstrates the correctness and reliability of the test results.

[0078] Example 3: Establishing a Predictive Model and Determining the Stage of Samples

[0079] This study established a methylation gene number prediction model using 50 early-stage, 50 mid-stage, and 50 late-stage esophageal cancer samples, and staged 80 samples.

[0080] For ease of description and distinction, this embodiment only describes the differences from Embodiment 2. Steps (1)-(5) in this embodiment also adopt the five steps of sample preservation, DNA preparation, sulfite modification, digital PCR, droplet reading and signal analysis in Embodiment 2, and will not be described again here.

[0081] (6) Detection revealed that as esophageal cancer progresses, more gene test results became positive, and the detection rate of esophageal cancer samples varied at different stages when different numbers of gene tests were used (e.g., Figure 2 (As shown in the figure). Specifically, using 1 gene test can identify 66% of early samples; using 2 gene tests can identify 100% of early samples and 4% of mid-stage samples; using 3 gene tests can identify 54% of mid-stage samples; using 4 gene tests can identify 96% of mid-stage samples and 8% of late-stage samples; using 5 gene tests can identify 100% of early samples and 90% of late-stage samples; and using 6 gene tests can identify 100% of late-stage samples.

[0082] Table 2. Detection of esophageal cancer samples at different time periods

[0083]

[0084] (7) Determine the sample period based on the number of positive genes detected, and plot ROC curves with FPR=FPR=FP / (FP+TN) as the x-axis and TPR=TP / (TP+FN) as the y-axis. Where:

[0085] FN represents the number of samples predicted for the target period but actually for other periods;

[0086] FP represents the number of samples that were predicted to be from other periods but actually occurred in the target period;

[0087] TN represents the number of samples where both the forecast and the actual number are the target period.

[0088] Thus, ROC curves corresponding to the prediction of early, intermediate, and late-stage esophageal cancer (e.g.) were plotted. Figure 10-12 ),from Figure 10-12 The AUC values ​​of the ROC curves corresponding to early prediction of esophageal cancer were 0.983, 0.967, and 0.934, respectively.

[0089] (8) Furthermore, by analyzing the test results of 50 early, 50 mid, and 50 late-stage samples in this embodiment, the correlation between different numbers of positive genes and esophageal cancer samples at different stages is shown in Table 3 below.

[0090] Table 3. Correlation between different numbers of positive genes and esophageal cancer samples from different time periods.

[0091]

[0092] When only two or fewer gene methylation tests are positive, the specificity of early-stage esophageal cancer samples is high, and we predict that the patient is in the early stage of esophageal cancer. When three to four gene methylation tests are positive, the specificity of mid-stage esophageal cancer samples is high, and we predict that the patient is in the mid-stage of esophageal cancer. When five to six gene methylation tests are positive, the specificity of late-stage esophageal cancer is high, and we predict that the patient is in the late stage of esophageal cancer. Under this standard, 80 samples were used for staging prediction, and the endoscopic gold standard test was used as a reference. The results are shown in Table 4 below.

[0093] Table 4. Staging of esophageal cancer samples

[0094]

[0095] As can be seen from Table 4, the predictions generated by this invention have small errors and high consistency.

[0096] In summary, this invention provides an esophageal cancer diagnostic kit based on the detection of multiple gene methylation levels. It features high sensitivity and specificity, and is convenient and quick to operate. By forming combinations of gene methylation, it enables early screening of esophageal cancer samples. Furthermore, the kit, combined with a digital PCR platform, leverages its advantages of absolute quantification, high batch-to-batch stability, and high sensitivity to accurately distinguish the relationship between sample gene methylation and esophageal cancer. Simultaneously, the use of fluorescently labeled probes for genotyping detection provides high specificity and is suitable for esophageal cancer patients at different stages. Therefore, it provides a convenient, rapid, and accurate detection method for early screening, diagnosis, and prognosis of esophageal cancer, possessing significant clinical value.

[0097] Furthermore, this invention can be used to establish methylation level assessment models and multi-gene prediction combinations, and applied to the simultaneous detection of multiple methylation sites in clinical samples; by identifying samples with Ct values ​​less than Cutoff values ​​as methylation positive, based on the methylation positivity of multiple sites, early-stage patients at risk of esophageal cancer can be screened, esophageal cancer patients at different stages can be distinguished, and the spread of esophageal cancer in other tissues can be understood.

[0098] Furthermore, it should be noted that all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

Claims

1. An esophageal cancer diagnostic kit based on the detection of multi-gene methylation levels, characterized in that: The esophageal cancer gene methylation detection sites detected by the kit include ZNF154, RNF126, C2ORF27A, OTOP2, TM4SF19, and P16, with sequences shown in SEQ ID NO. 1-12. The kit also includes a characteristic detection reagent, which comprises a dye-based quantitative PCR premix and the following primer and probe combinations: 1) Primer and probe combinations for ZNF154 methylation detection, including Mut-specific primers as shown in SEQ ID NO.13, Mut-specific primers as shown in SEQ ID NO.14, Mut-specific probes as shown in SEQ ID NO.25, Wt-specific primers as shown in SEQ ID NO.31, Wt-specific primers as shown in SEQ ID NO.32, and Wt-specific probes as shown in SEQ ID NO.43; 2) Primer and probe combinations for RNF126 methylation detection, including Mut-specific primers as shown in SEQ ID NO.15, Mut-specific primers as shown in SEQ ID NO.16, Mut-specific probes as shown in SEQ ID NO.26, Wt-specific primers as shown in SEQ ID NO.33, Wt-specific primers as shown in SEQ ID NO.34, and Wt-specific probes as shown in SEQ ID NO.44; 3) Primer and probe combinations for C2ORF27A methylation detection, including Mut-specific primers as shown in SEQ ID NO.17, Mut-specific primers as shown in SEQ ID NO.18, Mut-specific probes as shown in SEQ ID NO.27, Wt-specific primers as shown in SEQ ID NO.35, Wt-specific primers as shown in SEQ ID NO.36, and Wt-specific probes as shown in SEQ ID NO.45; 4) Primer and probe combinations for OTOP2 methylation detection, including Mut-specific primers as shown in SEQ ID NO.19, Mut-specific primers as shown in SEQ ID NO.20, Mut-specific probes as shown in SEQ ID NO.28, Wt-specific primers as shown in SEQ ID NO.37, Wt-specific primers as shown in SEQ ID NO.38, and Wt-specific probes as shown in SEQ ID NO.46; 5) Primer and probe combinations for TM4SF19 methylation detection, including Mut-specific primers as shown in SEQ ID NO.21, Mut-specific primers as shown in SEQ ID NO.22, Mut-specific probes as shown in SEQ ID NO.29, Wt-specific primers as shown in SEQ ID NO.39, Wt-specific primers as shown in SEQ ID NO.40, and Wt-specific probes as shown in SEQ ID NO.47; 6) Primer and probe combinations for P16 methylation detection, including Mut-specific primers as shown in SEQ ID NO.23, Mut-specific primers as shown in SEQ ID NO.24, Mut-specific probes as shown in SEQ ID NO.30, Wt-specific primers as shown in SEQ ID NO.41, Wt-specific primers as shown in SEQ ID NO.42, and Wt-specific probes as shown in SEQ ID NO.

48.

2. The esophageal cancer diagnostic kit based on multi-gene methylation level detection according to claim 1, characterized in that: The test sample type for this kit is an ex vivo blood sample.

3. The esophageal cancer diagnostic kit based on multi-gene methylation level detection according to claim 1, characterized in that: The 5' end of each of the specific probes contains a fluorescent group, which includes FAM and VIC.

4. The esophageal cancer diagnostic kit based on multi-gene methylation level detection according to claim 3, characterized in that: The fluorescent group of the Mut-specific probe is FAM, and the fluorescent group of the Wt-specific probe is VIC.

5. The esophageal cancer diagnostic kit based on multi-gene methylation level detection according to claim 1, characterized in that: The 3' end of each of the specific probes contains a quenching group, which is MGB.

6. The esophageal cancer diagnostic kit based on multi-gene methylation level detection according to claim 1, characterized in that: The reaction system of the kit is as follows: 1 μL of treated DNA (sulfurized template), 2.4 μL of 10 μM upstream and downstream primers, 0.75 μL of 10 μM Mut / Wt probe, 15 μL of dye-based quantitative PCR premix, and 8.45 μL of water are mixed to form a homogeneous 30 μL system.

7. The esophageal cancer diagnostic kit based on multi-gene methylation level detection according to claim 1, characterized in that: The PCR reaction conditions for the kit are as follows: 。

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